Ordered distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells revealed by freeze-fracture following treatment with cholesterol probes.код для вставкиСкачать
THE ANATOMICAL RECORD 232:385-392 (1992) Ordered Distribution of Membrane-Associated Dense Plaques in Intact Quail Gizzard Smooth Muscle Cells Revealed by Freeze-Fracture Following Treatment With Cholesterol Probes ELAINE C. DAVIS AND RICHARD R. SHIVERS Cell Sciences Laboratories, Department of Zoology, University of Western Ontario, London, Ontario, Canada ABSTRACT The surface distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells was investigated by freeze-fracture. Replicas of fractured smooth muscle cell plasma membrane showed caveola-free regions with few intramembrane particles, interspersed with caveola-populated areas with a higher intramembrane particle density. Electron microscopy of thin sections of quail gizzard smooth muscle revealed the regions free of caveolae to be occupied by membrane-associated dense plaques; anchoring sites for the contractile filaments of the cell. Demarcation between the caveola-populated and caveolafree regions on the relicated intramembrane surface was not clear and thus provided little information concerning the distribution of dense plaque sites. However, treatment of the smooth muscle tissue with the cholesterol-binding agents filipin or tomatin prior to freeze-fracture allowed the dense plaque sites to be easily observed as the sites remained free of the membrane deformations characteristic of these agents. The dense plaque sites consist of caveola-free oval areas juxtaposed in regular bands that traverse the long axis of the cell. The dense plaque sites on the freeze-fracture replica were confirmed by electron microscopy of thin sections of filipin-treated quail gizzard smooth muscle cells, which showed the plasma membrane associated with the dense plaques to be unaffected by the actions of filipin, whereas that of the caveola-populated region was severely deformed. The observations presented in this study provide evidence for a highly ordered distribution of dense plaques at the cell surface of intact quail gizzard smooth muscle cells and thus corroborate existing evidence for a n organized substructure of smooth muscle cells. The existence of distinct contractile units in smooth muscle cells remains uncertain. Thin filaments insert into cytoplasmic dense bodies that appear randomly distributed throughout the cell (Hanson and Lowy, 1961; Szent-Gyorgyi et al., 1971; Bois, 1973). Decoration of these filaments with myosin S-1 revealed their opposite polarity on either side of a given dense body, thereby establishing the role of the cytoplasmic dense body a s a functional equivalent to the Z-disc of vertebrate striated muscle (Bond and Somlyo, 1982). A series of “sarcomeres,” similar to those of striated muscle, may therefore exist within the smooth muscle cell, consisting of thin filament arrays, thick filaments, and cytoplasmic dense bodies, thus constituting a contractile unit (Bois, 1973; Sobieszek, 1973; Bond and Somylo, 1982). Clearly, the terminal thin filament array of such a contractile unit must be securely anchored to the cell membrane to be functional in the tramsmission of tension (Marston and Smith, 1985). These anchoring sites have been identified as dense plaques bound to the inner surface of the smooth muscle cell membrane (Bois, 1973; Somylo, 1980; Hunt, 1981). An organized three-dimensional arrangement of the contractile apparatus of smooth muscle cells has yet to 0 1992 WILEY-LISS, INC be established, as the spatial distribution of the contractile elements is difficult to ascertain. The demonstration of regular fibrillar patterns within intact (Rosenbluth, 1965) and isolated (Small, 1974) smooth muscle cells has indicated some degree of order within the cell. The fibrils, thought to represent contractile units, are oriented oblique to the long axis of the cell (Rosenbluth, 1965; Small, 1974) and become progressively more angled upon contraction (Fay and Delise, 1973; Small, 1974; Fisher and Bagby, 1977). In addition, phase contrast movies made of isolated cells reveal a helical or corkscrew appearance of the cells that accompanies contractile filament rearrangement during cell contraction (Fisher and Bagby, 1977). To account for these observations, Small (1977) and Fisher and Bagby (1977) proposed similar models for the organization of the contractile apparatus of smooth muscle cells. The models depict contractile units arranged Received July 3, 1991; accepted September 20, 1991. Address reprint requests to Elaine C. Davis, Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7. E.C. DAVIS AND R.R. SHIVERS 386 in a common spiral about the long axis of the cell. Such a n arrangement indirectly suggests a highly ordered helical distribution of membrane-associated dense plaques a t the cell surface; however, to date, direct evidence of such is lacking. In conventional electron microscopy, membrane-associated dense plaques are easily observed. However, the overall spatial distribution of the plaque sites is difficult to perceive. In the present study, freeze-fracture techniques have been used to investigate the organization of dense plaque sites over the surface of intact quail gizzard smooth muscle cells. As these sites present no intramembrane specialization recognizable by freeze-fracture, the cholesterol binding agents filipin and tomatin were used. These agents react specifically with membrane cholesterols and other related 3-beta-hydroxysterols to produce distinctive surface deformations easily recognized on freeze-fracture replicas (Elias et al., 1979; Severs and Robenek, 1983). Filipin and tomatin bind in a heterogenous manner to smooth muscle cell membrane sterols (Montesano, 1979; Severs and Simons, 1983) and indirectly demarcate the dense plaque sites on the replicated intramembrane surface. Results from the present study support existing evidence of dense plaque organization observed in isolated avian smooth muscle cells (Draeger e t al., 1989). Because these investigators were not able to demonstrate a similar organization in intact avian smooth muscle cells and because the organization observed in the isolated avian cells is in direct contrast to that observed in other isolated smooth muscle cell types (Small, 1985), i t is important to establish that a n ordered transverse arrangement of dense plaque sites indeed exists in intact quail gizzard smooth muscle cells by methods that do not involve disruption of the cells by isolation techniques. Although the three-dimensional organization of the dense plaque sites and their associated intracellular filaments remains to be determined, the organized dense plaque sites observed in the present study highly suggests that some degree of ordered substructure must exist within smooth muscle cells. (Heuser and Reese, 1973). The following day, the tissue was rinsed in sodium acetate, dehydrated in a graded series of methanol to propylene oxide, infiltrated in mixtures of propylene oxide and British Araldite (Polysciences Inc., Warrington, PA), and embedded in fresh Araldite. Silver sections were cut with glass knives on a Reichert Om-U2 ultramicrotome, picked up on bare 200-mesh grids, and stained with methanolic uranyl acetate (Franc et al., 1984) and lead citrate (Reynolds, 1963). Pieces of tissue for freeze-fracture were initially fixed for 1 hour in 3% glutaraldehyde in 0.1 M sodium cacodylate prior to being transferred to fresh fixative containing either filipin (Sigma Chemical Co., St. Louis) a t a concentration of 400 pg/ml, or tomatin (Sigma Chemical Co.) a t 150 pg/ml (Severs and Robenek, 1983). Both solutions also contained 1% dimethyl sulfoxide (DMSO). A control sample containing only fresh fixative and 1% DMSO was also prepared. All three samples were left 24 hours a t room temperature in lightproof vials, then washed with several changes of sodium cacodylate buffer. At this point, part of the filipin-treated tissue and part of the control tissue were removed and processed for thin section electron microscopy a s previously described. The remainder of the three samples was placed in cacodylatebuffered 30% glycerol (pH 7.35) overnight a t 4 "C. The following day, small pieces of tissue were placed on gold specimen discs (Balzers, Liechtenstein), suspended in a drop of glycerol, and frozen in a slurry of Freon-22 cooled with liquid nitrogen. The tissue was freeze-fractured at -112 "C in a Balzers BAF-301 Freeze-Etch Unit and shadowed with a thin layer of platinum and carbon according to the method of Shivers and Brightman (1976). The replicas were cleaned in household bleach, washed in three changes of filtered distilled water, and picked up on bare 200-mesh copper grids. The silver thin sections and platinum replicas were examined in a Philips 201 transmission electron microscope operating at a n accelerating voltage of 60 kV. MATERIALS AND METHODS The contractile machinery of the gizzard smooth muscle cell consists of thin filaments, thick filaments, and oval-shape cytoplasmic dense bodies; all oriented parallel t o the long axis of the cell in a n apparent random manner (Figs. 1,2). At the plasma membrane, bands of caveolae alternate with membrane-associated Adult Japanese quails (Coturnix coturnix japonica) were obtained from a n inbred colony maintained in the Animal Care Facility of the Department of Zoology a t the University of Western Ontario (London, Ont.). The animals were decapitated and the gizzards immediately removed. The thick muscle was dissected from the gizzard and cut into 1 mm3 pieces for electron microscopy and freeze-fracture. Immediately following dissection, tissue pieces for thin sections were fixed for 2 hours a t room temperature in 3%glutaraldehyde buffered to pH 7.35 with 0.1 M sodium cacodylate. The tissue was then rinsed in several changes of cacodylate buffer and postfixed for 1 1/2 hours in 1%osmium tetroxide buffered with 0.1 M sodium cacodylate. After osmication, the tissue was rinsed and treated for 1 hour with 2% tannic acid in cacodylate buffer to enhance electron density and contrast of cell surface and intracellular structures (Wagner, 1976). Following a thorough wash in 0.1 M sodium acetate (pH 5.21, the tissue was stained en bloc with aqueous uranyl acetate and left overnight a t 4°C RESULTS Fig. 1. Cross section through several quail gizzard smooth muscle cells showing general ultrastructural features. Cytoplasmic dense bodies (CDB) appear randomly distributed among the contractile filaments that fill much of the cell interior. At the cell surface, alternating regions of membrane-associated dense plaques (MADP) and bands of caveolae (CAV) are observed. No regular pattern of distribution of these membrane specializations can be discerned. (M) mitochondria, (N) nucleus. x 23,430;bar = 0.5 Km. Fig. 2. Longitudinal section through adjacent edges of two quail gizzard smooth muscle cells showing a regular distribution of membrane-associated dense plaques (MADP) and caveolar bands ICAV) along the cell membrane. The pattern of distribution is emphasized by the slight contraction of the cells, which causes the cell surface to become scalloped. Note the contractile filaments of the cell which anchor into the membrane-associated dense plaques (arrows). X22,750; bar = 0.5 Km. Figs. I , 2 Figs. 3-6 DENSE PLAQUE DISTRIBUTION IN SMOOTH MUSCLE CELLS dense plaques. The dense plaques are thin, not extending far into the cytoplasm, and are penetrated by the termini of the intracellular filaments (Fig. 2). In crosssectioned cells, the dense plaques appear to vary considerably in size with no discernable organization (Fig. 1). In cells sectioned longitudinally, the dense plaques often appear to have some regularity t o their distribution (Fig. 2). In control tissue, treated with DMSO only in the fixative, the plasma membrane associated with the dense plaques is smooth and normal in appearance. Similarly, individual caveolae are easily recognized within the caveolar bands and are clearly delimited by the plasma membrane (Fig. 3). In contrast, treatment of the tissue with fixative containing DMSO and filipin causes the caveolar bands to be almost indistinguishable, and individual caveolae cannot be recognized due to severe deformation of the plasma membrane. The plasma membrane associated with the dense plaques, however, is undisturbed by the treatment and remains smooth (Fig. 4). Freeze-fracture replicas of the quail gizzard smooth muscle cell intramembrane surface show extensive areas with few intramembrane particles and no caveolar ostia, surrounded by regions of intramembrane surface with a considerably greater density of particles and numerous caveolar ostia (Fig. 5). No recognizable specializations on the replicated surface identify the dense plaque sites. However, since thin sections show the re- Fig. 3. High magnification of adjacent edges of two quail gizzard smooth muscle cells treated with DMSO only in the fixative. At the surface of the upper cell, a band of caveolae is observed (CAV). Note that the plasma membrane delimiting each caveola is smooth and well defined. The opposing membrane shows a membrane-associated dense plaque (MADP). Extracellular to the plaque, the basement membrane appears especially electron-dense (arrows). (GJ) Gap junction. ~ 8 8 , 0 6 0bar ; = 0.2 pm. Fig. 4. High magnification of adjacent edges of two quail gizzard smooth muscle cells treated with filipin and DMSO in the fixative. The plasma membrane onto which the membrane-associated dense plaque (MADP) is affixed is smooth and unaffected by the experimental treatment (arrows). In contrast, the membrane of the caveolae within a caveolar band (CAV) is highly sensitive to the cholesterol binding actions of filipin. Individual caveolae are almost indistinguishable due to the severe deformation of the membrane by the formation of filipin-cholesterol complexes. Intracellular structures, such as thin filaments (tF), thick filaments (TF), and cytoplasmic dense bodies (CDB), are unaffected. x 88,060; bar = 0.2 pm. Fig. 5. Freeze-fracture replica of the intramembrane surface (PF fracture face) of a quail gizzard smooth muscle cell treated with DMSO only in the fixative. Two regions of the intramembrane surface are distinguishable; one, heavily populated by intramembrane particles that surround numerous caveolar ostia (GO), is the location of the caveolar bands, whereas the other, which contains few intramembrane particles and no caveolar ostia, is thought to reflect the underlying membrane-associated dense plaques (MADP).Note that the border between the two regions is not well defined. (GJ) Gap junction. ~ 6 1 , 6 0 0bar ; = 0.2 pm. Fig. 6. Freeze-fracture replica of the intramembrane surface (PF fracture face) of a quail gizzard smooth muscle cell treated with filipin and DMSO in the fixative. Filipin-cholesterol complexes (FC) appear on the intramembrane surface in distinct regions among the caveolar ostia ((20). In contrast, the regions of the intramembrane surface that correspond to underlying membrane-associated dense plaques (MADP) show no deformation by filipin-cholesterolcomplexes, as similarly seen in Figure 4. x 61,600; bar = 0.2 pm. 389 gions of caveola-free cell membrane to be occupied by dense plaques (Figs. 1,2), it is likely that the caveolafree areas on the replicated intramembrane surface represent the plaque sites. Despite this plausible localization, the lack of a distinct boundry between the caveola-free and caveola-populated areas make any organization of the plaque sites inconspicuous. In an attempt to demarcate the dense plaque sites on the replicated intramembrane surface, the sterolbinding agents filipin and tomatin were used. The antibiotic filipin is clearly selective in its binding pattern. Filipin-sterol complexes appear in large numbers among the caveolar ostia, whereas only a few complexes are evident in the caveola-free regions (Fig. 6). These caveola-free areas, which are devoid of filipinsterol complexes, clearly reflect the location of the underlying dense plaques as corroborated by the thin section observations of filipin treated tissue (Fig. 4).In addition, the dense plaque areas seen on the replicated surface are roughly oval in shape and appear consistent in size with dense plaques observed in thin section. At low magnification, the heterogenous distribution of filipin-sterol complexes reveals a distinct pattern of the complex-free dense plaque sites (Fig. 7). Individual plaque sites are juxtaposed in regularly oriented bands that traverse the long axis of the cell. In some instances, the plaque sites free of filipin-sterol complexes appear to merge, emphasizing the ordered nature of the sites into regular bands that traverse the long axis of the cell (Fig. 7). Treatment of the smooth muscle tissue with tomatin produces similar results to those obtained with filipin. Hemitubular grooves, characteristic of tomatin treatment, severely deform the intramembrane surface except in distinct oval areas where the surface appears less disrupted (Fig. 8). These areas are oriented side by side in regular bands that traverse the long axis of the cell in an identical manner to the plaque sites delimited by filipin (Fig. 8). Although the plaque sites are evident, tomatin does cause some deformation of the plaque site membrane, thus making it less valuable as an agent to demarcate membrane specializations. DISCUSSION The heterogenous distribution of membrane deformities resulting from treatment of quail gizzard smooth muscle with filipin or tomatin has provided an indirect opportunity to study the spatial distribution of membrane-associated dense plaques over the cell surface in intact cells. In the present study, thin sections and platinum replicas of filipin-treated smooth muscle cells show areas of the cell membrane devoid of filipin-sterol complexes that correspond to the membrane-associated dense plaques. These results are consistent with similar studies of filipin-treated smooth muscle where filipin-induced deformations were abundant in regions of the sarcolemma containing closely packed caveolae while absent from adjacent caveola-free areas (Montesano, 1979; Severs and Simons, 1983, 1986). Early research suggested that this heterogenous distribution of filipin-sterol complexes reflected a similar heterogenous distribution of cholesterol and 3-beta-hydroxysterols in the smooth muscle cell membrane (Montesano, 1979). However, the resistance of caveola-free areas to deformation by filipin was shown to be a false 390 E.C. DAVIS AND R.R. SHIVERS Fig. 7. Low magnification of a freeze-fracture replica of the intramembrane surface of a filipin-treated quail gizzard smooth muscle cell showing the organized distribution of membrane-associated dense plaques as revealed by their resistance to deformation by filipin. The oval-shape dense plaques (bracketed)are organized juxtaposed in regular hands that traverse the long axis of the cell. x 10,665; bar = 1.0 Fm. Fig. 8. Freeze-fracture replica of the intramembrane surface of a quail gizzard smooth muscle cell treated with tomatin in the fixative. Tomatin complexes with membrane cholesterols to produce distinct hemitubular deformations (arrows). As with filipin, regions of the intramembrane surface associated with dense plaques are less affected by the treatment, and thus a similar pattern of individual plaques (bracketed) organized in regular transverse bands is revealed. x 16,590; bar = 1.0 Fm. negative cytochemical result since treatment of the smooth muscle tissue with the cholesterol binding agent tomatin produced characteristic hemitubular tomatin-sterol complexes in the caveola-free areas (Severs and Simons, 1983, 1986). The fact that filipin cannot bind to membrane cholesterols in the caveola-free regions provides further evidence that these regions do indeed correspond to the underlying membrane-associated dense plaques since evidence from several studies suggests that membrane specializations can act by various mechanisms to physically interfere with complex formation (Severs et al., 1981; Severs and Robenek, 1983; Berdan and Shivers, 1985). The underlying proteins of the dense plaque attached to the cytoplasmic surface of the cell membrane may prevent membrane deformation due to increased membrane rigidity (Steer et al., 1984; Severs and Simons, 1986).In addition, both the small filaments that extend from the cell membrane at the dense plaque site and the thickened lamina densa may inhibit filipin-sterol complex formation by preventing penetration of the agent to the membrane. Similar filaments radiate from hemidesmosomes and are thought to be responsible, a t least in part, for the lack of filipin binding in that membrane domain (Berdan and Shivers, 1985). The resultant pattern of membrane-associated dense plaques observed following filipin and tomatin treatment provides evidence for some degree of order to the underlying substructure of the smooth muscle cell. This is the first demonstration that membrane-associ- DENSE PLAQUE DISTRIBUTION IN SMOOTH MUSCLE CELLS 391 ated dense plaques are organized juxtaposed in regular thus remains elusive due to contradicting results. Intransverse bands across the longitudinal axis of intact creasing evidence suggests that one model may not exsmooth muscle cells. In contrast, the freeze-fractured ist to encompass all smooth muscle cell types. In intramembrane surface of smooth muscle cells from the smooth muscle cells of the vena cava as compared to rabbit vena cava was shown to consist of longitudinally those of the aorta and pulmonary artery, a difference in oriented caveola-free bands, associated with the under- the size, shape, and distribution of membrane-associlying dense plaques, that alternate with bands of ca- ated dense plaques is reflected in differences observed veolae (Severs and Simons, 1986). The longitudinal in caveolar banding patterns and reaction to filipin banding pattern was accentuated by the treatment of treatment (Severs and Simons, 1986). In addition, the the tissue with filipin, which clearly showed the filipin- aortic smooth muscle cells appears to vary from the sensitive bands of caveolae to extend 20 pm or more femoral artery and gizzard smooth muscle cells in the along the cell length. A similar longitudinal arrange- distribution of F-actin, alpha-actinin, and filamin (Fument of bands of caveolae and caveola-free regions was jimoto and Ogawa, 1988). The femoral artery smooth observed upon freeze-fracture of strips of smooth mus- muscle cells, in turn, differ from those of the gizzard in cle from the rat large intestine (Montesano, 1979) and the number of cytoplasmic dense bodies and the shape of the guinea pig taeniae coli (Gabella and Blundell, of the membrane-associated dense plaques along the 1978).These results are consistent with those revealed cell surface (Gabella, 1981). Thus the dissimilar ultraby immunofluorescence microscopy that localize vincu- structural features of vascular and visceral smooth lin, a protein specific to membrane-associated dense muscle cells, and even venous and arterial smooth plaques, in parallel coaxial bands on the surface of sev- muscle cells, may actually reflect a basic difference in eral types of isolated vertebrate smooth muscle cells the contractile machinery organization as a result of (Small, 1985). In other types of smooth muscle, such as the different mechanical demands imposed upon the from the rabbit aorta and pulmonary artery, the cave- various smooth muscle cell types (Severs and Simons, olae formed irregularly distributed aggregations and 1986; Fujimoto and Ogawa, 1988). since large areas of fractured surface were not obIn the present study, the regular transverse distritained, any pattern of distribution, either transverse or bution of membrane-associated dense plaques supports longitudinal, was not evident (Severs and Simons, existing evidence for an organized substructure for 1986). smooth muscle cells. Although the results presented in Although the freeze-fracture evidence thus far seems this study may be in contrast to others, increasing evto favor a longitudinal arrangement of membrane-as- idence suggests that each type of smooth muscle must sociated dense plaques along most smooth muscle cell be investigated separately before overall conclusions surfaces, several studies have demonstrated a trans- and generalization can be considered. verse organization of various intracellular components LITERATURE CITED of the contractile apparatus. In isolated guinea pig taenia coli smooth muscle cells, phosphorylated myosin Bennett, J.P., R.A. Cross, J. Kendrick-Jones, and A.G. Weeds 1988 was localized by immunofluorescence into a series of Spatial pattern of myosin phosphorylation in contracting smooth muscle cells: Evidence for contractile zones. J. 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