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Ordered distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells revealed by freeze-fracture following treatment with cholesterol probes.

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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
Cell Sciences Laboratories, Department of Zoology, University of Western Ontario, London,
Ontario, Canada
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,
An organized three-dimensional arrangement of the
contractile apparatus of smooth muscle cells has yet to
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
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
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,
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
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.
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
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 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.
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.
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
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
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-
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
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
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