/. Embryol. exp. Morph. Vol. 72, pp. 53-69, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Increased adhesiveness at sites of high
acetylcholine receptor density on embryonic
amphibian muscle cells cultured without
nerve
ByF. MOODY-CORBETT 1 AND M. W. COHEN 2
From the Department of Physiology, McGill University, Montreal, Quebec
SUMMARY
In culture, myotomal muscle cells from Xenopus laevis embryos develop discrete patches of
high acetylcholine receptor (AChR) density. To examine the relative adhesiveness of these
sites, muscle cells having AChR patches on their lower surface (apposed to the culture dish)
were identified and were then treated with dibucaine or potassium-Ringer in order to cause
the cells to round up. More than 90% of these cells remained attached at an AChR patch
after rounding up, and this was the case even when the cells had a single patch on their lower
surface. When the cells were torn away from the culture dish by mechanical agitation, small
cellular fragments still remained firmly attached to the dish and many of these fragments
contained an AChR patch. It is concluded that AChR patches on the lower surface of the cell
are often located at sites of increased adhesiveness to the culture dish. The findings are
discussed in terms of the formation and maintenance of AChR patches.
INTRODUCTION
When embryonic muscle cells are cultured in the absence of nerve they
develop surface patches of high acetylcholine receptor (AChR) density (Vogel,
Sytkowski & Nirenberg, 1972; Fischbach & Cohen, 1973; Hartzell & Fambrough, 1973; Axelrod et al 1976; Anderson, Cohen & Zorychta, 1977;
Powell & Friedman, 1977). This is the case even if the muscle cells have never
been previously contacted by nerve (Bekoff & Betz, 1976; Moody-Corbett &
Cohen, 1981). The density of AChRs within these patches can be an order of
magnitude greater than elsewhere on the muscle cell and approaches that seen at
the adult neuromuscular junction (Sytkowski, Vogel & Nirenberg, 1973;
Anderson et al. 1977; Land, Podleski, Salpeter & Salpeter, 1977). Many of the
AChR patches are also relatively stable and survive in a fixed position for several
days (Anderson & Cohen, 1977; Frank & Fischbach, 1979; Moody-Corbett &
Cohen, 1982). Embryonic muscle cells thus have a means of accumulating AChRs
1
Author's present address: Department of Physiology, Tufts University, Boston,
Massachusetts 02111, U.S.A.
2
Author's address: Department of Physiology, McGill University, 3655 Drummond Street,
Montreal, Quebec, Canada, H3G1Y6.
53
54
F. MOODY-CORBETT AND M. W. COHEN
into discrete patches and of maintaining these accumulations in the absence of
innervation.
The mechanisms which participate in the development and maintenance of
AChR patches and their relevance to the accumulation of AChRs at the developing neuromuscular junction are currently the focus of considerable investigation.
Evidence has been obtained which indicates that the AChRs within AChR
patches are much less mobile than those present at lower density elsewhere on
the cell (Axelrod et al. 1976). AChRs at the neuromuscular junction also
appear to be immobile (Axelrod et al. 1976; see also Fambrough, 1979). These
findings have led to the notion that the formation and/or maintenance of
discrete sites of high AChR density is dependent upon an anchorage of the
AChRs at those sites (Axelrod et al. 1976; see also Anderson & Cohen, 1977;
Orida & Poo, 1978; Bloch, 1979). Further studies have suggested that the
AChRs may be anchored by components of the basal lamina on the external
surface of the plasma membrane (Burden, Sargent & McMahan, 1979; Heuser
& Salpeter, 1979; Heuser, 1980; McMahan, Edgington & Kuffler, 1980a;
McMahan, Sargent, Rubin & Burden, 1980&) and by intracellular components
which interact with the inner surface of the plasma membrane (Ellisman, Rash,
Staehelin & Porter, 1976; Fertuck & Salpeter, 1976; Heuser & Salpeter, 1979;
Bloch & Geiger, 1980; Heuser, 1980; Cartaud et al. 1981; Couteaux, 1981;
Froehner et al. 1981; Hall, Lubit & Schwartz, 1981). In culture the occurrence
of some AChR patches may also be related to direct contact with an apposing
surface (Axelrod, 1981; Peng, Cheng & Luther, 1981).
The formation and survival of discrete accumulations of other surface receptors, such as receptors for lectins and immunoglobulins, have been found to be
markedly affected by agents such as colchicine and cytochalasin B which
disrupt microtubules and microfilaments, thereby implicating these intracellular
structures in the organization of the cell surface (for review, see Edelman, 1976).
By contrast studies on cultures of rat myotubes have indicated that the occurrence of AChR patches is only slightly reduced by colchicine and is unaffected
by cytochalasin B (Axelrod, Ravdin & Podleski, 1978; Bloch, 1979). Nor does
colchicine appear to prevent the development of AChR patches on embryonic
chick muscle cells in culture (Fukuda, Henkart, Fischbach & Smith, 1976).
The initial aim of the present study was to determine whether the AChR
patches on embryonic Xenopus muscle cells in culture are also insensitive to
treatment with colchicine, cytochalasin B and dibucaine. Dibucaine was
chosen because of its reported effectiveness in disrupting caps of immunoglobulin receptors presumably through disruption of both microtubules and microfilaments (Poste, Papahadjopoulous, Jacobson & Vail, 1975a; Poste, Papahadjopoulous & Nicolson, 1975Z?). During the course of our experiments we
found that high concentrations of dibucaine caused the muscle cells to peel off
the culture dish almost completely and round up. Many of these rounded up
cells remained attached at the site of an AChR patch suggesting that this region
Increased adhesiveness at AChR patches
55
is more strongly adhesive than other areas of the cell in contact with the culture
dish.
The present study examines the latter effect in more detail and also includes a
brief description of the survival of AChR patches in cultures treated with
colchicine, cytochalasin B and lower concentrations of dibucaine. Our experiments confirm that AChR patches on Xenopus muscle cells often occur at sites
of increased adhesion to the culture dish. This finding, in line with recent
studies which indicate that AChR patches on rat muscle cells in culture often
occur at sites of close contact with the culture dish (Axelrod, 1980, 1981;
Bloch & Geiger, 1980), extends the list of similarities between AChR patches on
Xenopus muscle cells in culture and the postsynaptic membrane at the neuromuscular junction (see Discussion).
METHODS
Muscle cultures were prepared as described previously (Anderson et al. 1977;
Moody-Corbett & Cohen, 1981). All dissections were done under sterile conditions. Briefly, the myotomes of stage-22 to -24 Xenopus laevis embryos (Nieuwkoop & Faber, 1967) were isolated from surrounding tissue with the aid of
collagenase and further dissociated into single cells in a calcium-magnesiumfree solution of trypsin and EDTA. The muscle cells were added to culture
chambers containing plating medium (67 % L-l 5 and 5 % dialysed horse serum).
One day later the medium was replaced with maintenance medium (67% L-l5
and 0-2/£g/ml Holmes' a-1-protein). All components of the culture medium
were purchased from Grand Island Biological Co. of Canada Ltd. Sealed
culture chambers were constructed as described by Anderson et al. (1977), their
floor consisting of a collagen-coated glass coverslip. Cultures of Xenopus
sympathetic and dorsal root ganglia were also prepared as previously described
(Cohen &Weldon, 1980).
Sites of high AChR density were stained with a-bungarotoxin conjugated
with tetramethylrhodamine (Anderson & Cohen, 1974; Anderson et al. 1977;
Cohen & Weldon, 1980). For microscopy, the sealed living cultures were
inverted and viewed with phase-contrast and fluorescence optics. Fine focusing
readily distinguished AChR patches on the lower surface of cells (the surface
apposed to the floor of the culture dish) from those on the upper surface. By
photographing specific cells and utilizing the coordinates of the microscope
stage the same cells could be followed before and after drug treatment. When
required the cultures were restained in the presence of the drug, in order to
keep the AChR patches brightly fluorescent. In experiments where successive
observations were made on the same culture different portions of the culture
were evaluated in order to reduce the chances of artifactual loss of AChR
patches by bleaching.
The effects of dibucaine and colchicine were examined by dissolving the
t
\
Fig. 1. Phase-contrast and corresponding fluorescence views of a muscle cell in a 4-day-old culture before (A) and after (B)
treatment with 0-2 mg/ml dibucaine. Two of the cell processes have ACh patches on the lower surface. After treatment with
dibucaine the muscle cell rounded up but remained attached at one of the patches. Scale bar is 20 /im.
" X,
m
Z
n
o
z
a
w
H
H
o
o
I
o
o
o
ON
Fig. 2. Phase-contrast and corresponding fluorescence views of portions of two muscle cells in a 5-day-old culture
before (A) and after (B) treatment with 0-5 mg/ml dibucaine. Each muscle cell had an AChR patch on its
lower surface. After the dibucaine treatment the cells rounded up (not shown) and further agitation caused the cells to
tear away from the dish. Only a small fragment (arrow) of one of the cells remained attached and this fragment
contained one of the AChR patches. Scale bar is 20 (im.
s
SiSi
I
5
58
F. MOODY-CORBETT AND M. W. COHEN
Table 1. Percentage of muscle cells which remained attached at an AChR patch
after rounding-up
Cells with AChR patches on their lower surface were first identified and photographed. Each of the selected cells had at least one brightly stained and relatively
large AChR patch (greatest dimension: 10-25 fim). The cells were rephotographed
after treatment with dibucaine or potassium-Ringer. The data include only those
cases where a major portion of the cell rounded up (see Figs. 1-4.)
Treatment
Dibucaine
Dibucaine
Potassium-Ringer
Category of cell
Rounded cells
attached at an
AChR patch (%)
More than one AChR patch on lower surface
One AChR patch on lower surface
One AChR patch on lower surface
93 (40)
93 (42)
100 (17)
Numbers in parentheses indicate total number of cells.
drugs directly in maintenance medium. Cytochalasin B was first dissolved in
dimethylsulphoxide and then diluted 100-fold in maintenance medium. The
drugs used in these experiments were purchased from Sigma Chemical Co.
For some experiments the adhesiveness at AChR patches on the lower surface
was examined by treating the cultures with a potassium-Ringer solution
(lllmM-KCl, l-8mM-CaCl2, 10 mM-HEPES buffer at pH 7-3) instead of
dibucaine. In some cases cells were intentionally torn away from the floor of the
culture chamber by repeatedly passing a small air bubble across the cells after
inverting the sealed chamber. Most of the experiments on muscle cell adhesiveness were done on 2 to 5-day-old muscle cultures but similar results were
obtained in a few experiments with cultures as old as 10 days.
RESULTS
Increased adhesion at AChR patches
Previous studies on endothelial cells in culture have revealed that dibucaine
can cause the cells to round up and even detach from the culture dish (Nicolson
& Poste, 1976; Nicolson, Smith & Poste, 1976). Treatment of Xenopus muscle
cultures with a high concentration of dibucaine (0-2-1-0 mg/ml) also caused
the muscle cells to round up within 5-30 min. Usually the rounded-up cells
remained attached to the culture dish in a region which contained an AChR
patch on the lower surface (Fig. 1). In some cases, especially with further
agitation (see Methods), the rounded-up cells tore away completely from the
dish leaving behind small adherent cellular fragments which often contained
an AChR patch of approximately similar size (Fig. 2). As summarized in Table
1, in a sample of 40 identified cells which rounded up after exposure to dibucaine,
Increased adhesiveness at AChR patches
59
Table 2. Percentage of processes with or without lower surface AChR patches
which remained attached after the cell rounded up
Only processes which extended for at least 10 /im beyond the main margin of the
cell were considered (see Figs. 1 and 4). Processes without AChR patches were
considered attached if any part of the process was still in contact with the dish after
the cell rounded up. Processes with AChR patches were considered attached only if
an AChR patch was still in contact with the dish after the cell rounded up.
Processes attached (%)
Treatment
Dibucaine
Potassium-Ringer
Processes without AChR
patches
Processes with AChR
patches
8(119)
19 (16)
58(76)
80(10)
Numbers in parentheses indicate total number of processes. From the same
experiments as Table 1.
93 % either remained attached at an AChR patch (33 cells) or left behind a cell
fragment which contained an AChR patch (4 cells). Since AChR patches on the
lower surface of these muscle cells occupy less than 3 % of the surface area
(Moody-Corbett & Cohen, 1981) it is unlikely that these results are due to
chance. Rather they indicate that the region of the AChR patch is more resistant
to detachment by dibucaine than the remainder of the cell.
AChR patches on the lower surface of muscle cells in these cultures are
usually located at the ends of the cells or their processes (Anderson et al. 1977)
and one might imagine that these sites would be more adherent to the culture
dish even if they did not contain AChR patches. This possibility was examined
in two ways. Muscle cells having prominent processes, extending at least 10 /im
beyond the main border of the cell, were identified and their processes were
characterized according to whether or not they had AChR patches on their
lower surface (see Fig. 1). After treatment with dibucaine most of the processes
without AChR patches on their lower surface withdrew completely from the
culture dish; in only 8 % of the cases was some portion of a process still adherent
to the culture dish (Table 2). By contrast, the corresponding value for processes
with AChR patches on their lower surface was 58 % (Table 2). In a second set of
experiments the effect of dibucaine was examined on muscle cells which had
only a single AChR patch on their lower surface. Ninety-three percent of these
cells (n = 42) remained attached at the AChR patch (Table 1). As shown in
Fig. 3 this was the case even for cells whose AChR patch was located in the
central region of the cell. All of these observations indicate that regions of high
AChR density are preferentially more resistant to detachment by dibucaine.
Since in the above experiments the AChRs were stained with fluorescent
Fig. 3. Phase contrast and corresponding fluorescence views of two muscle cells in a 2-day-old culture before (A) and after (B)
treatment with 0-4 mg/ml dibucaine. Each cell had a single AChR patch on its lower surface. After treatment with
dibucaine the cells rounded up and remained attached in the region of the AChR patches, even in the case where the patch
was in the central region of the cell (lower muscle cell). Scale bar is 20/*m.
\
z
w
o
o
z
o
a
H
H
o
o
w
o
o
o
Fig. 4. Phase contrast and correspondingfluorescenceviews of a muscle cell in a 5-day-old culture before (A)
and after (B) the medium was changed to a Ringer solution with a high concentration of potassium. The
potassium-Ringer caused the muscle cell to contract and round up. However, the cell remained attached to the
dish at the two sites with AChR patches. Scale bar is 20 /*m.
"8
So
Ii
62
F. MOODY-CORBETT AND M. W. COHEN
toxin before exposure to dibucaine it was possible that the toxin somehow
increased adhesiveness at AChR patches. In order to examine this possibility
six cultures were first treated with dibucaine and were exposed to the fluorescent
toxin only after the cells had rounded up. Randomly chosen rounded-up cells
were then examined (mean sample size per culture: 52). In these six cultures
60 ±10% (mean and standard error) of the rounded-up cells were attached at
an AChR patch. In six control cultures (mean sample size per culture: 52)
which were stained first and then treated with dibucaine 59 ± 11 % of the
rounded-up cells were attached at an AChR patch. These results indicate that
prestaining with the fluorescent toxin did not enhance the adhesiveness of the
lower surface at AChR patches. The reason why the percentages in these
experiments are less than those in Table 1 may be related to the fact that some
muscle cells in these cultures have no AChR patches on their lower surface and
others have lower surface patches which are relatively small and/or faintly
stained. The cells in Table 1, however, were selected because they had relatively
large (greatest dimension: 10-25/tm) and brightly stained AChR patches on
their lower surface.
To check whether preferential attachment at AChR patches following dibucaine treatment is peculiar to this drug, additional experiments were made in
which muscle cells were caused to detach partially from the culture dish by
eliciting contraction with a Ringer solution containing a high concentration of
potassium (see Methods). Seventeen cells with single AChR patches on their
lower surface were followed in five cultures before and after changing the medium and, even though major portions of these cells detached from the culture
dish and rounded up, all of the cells remained attached at the region of the
AChR patch (Table 1). In addition, as with dibucaine treatment, muscle cell
processes with AChR patches were much less susceptible to detachment than
muscle cell processes without AChR patches (Table 2, Fig. 4). Finally, after
staining one muscle culture with fluorescent toxin almost all of the cells were
removed by extensive agitation so that only tightly adherent cell fragments
remained attached to the culture dish. The fragments were first identified with
phase-contrast optics and then examined for AChR stain. Fifty-three percent of
the fragments had AChR stain. In view of the fact that AChR patches occupy
less than 3 % of the lower cell surface (Moody-Corbett & Cohen, 1981), and the
possibility that some of the cell fragments were derived from non-muscle cells,
these findings further emphasize that AChR patches on the lower surface of the
muscle cell are sites where the cell is more firmly attached to the culture dish.
Survival of AChR patches in the presence of colchicine, cytochalasin B
and dibucaine
Colchicine and cytochalasin B, unlike dibucaine, did not cause muscle cells
to detach from the culture dish. Accordingly, it was possible to examine the
effects of these drugs on the survival of AChR patches. Dibucaine was also
Fig. 5. Phase contrast and correspondingfluorescenceviews of two muscle cells in a 4-day-old culture 16 h after the addition of
100/^g/ml colchicine to the medium. The upper cell had a normal appearance in phase contrast and also had an AChRtatch
on its lower surface. The lower cell appeared degenerated and had no AChR patches. Scale bar is 20/im.
ON
I
I
4
3
1 % Dimethylsulphoxide
4
3
3
1
Dibucaine
(100/*g/ml)
Cytochalasin B
(20/tg/ml)
Cytochalasin B
(100/*g/ml)
Colchicine
(100/tg/ml)
Treatment
Culture
age (day)
35
60
56
60
60
100
25
25
20
25
25
19
60
100
f Oh
1 16 h
L 2 days
16 h
16 h
2 days
" Oh
54
98
93
78
90
85
100
84
100
100
100
89
97
94
(%)
11*
No. of
patches
per cell
0
1
7
60
0
100
0
0
5
0
0
6
0
0
cells
No. of
1-5*
1-8*
N.D.
N.D.
N.D.
2-6 ±0-2
3h
21 ±0-3
6h
2-4 ±0-3
" Oh
3-3 + 0-3
3h
3-2±O-3
.11 h
2-3 ±0-4
16 h
N.D.
N.D.
2 days
N.D. denotes cases where the relevant values were not determined.
No. of
cells
Time after
adding drug
Cells with
AChR patches
Normal
No. of
patches
per cell
_
—
0-3
—
—
0
—
—
1-2 ±0-7
—
—
2-8±0-8
—
—
Cells with
AChR patches
(%)
_
0
29
0
—
0
—
—
40
—
—
100
—
—
Deteriorated
Bracketed data were obtained from the same culture. Values with an asterisk (*) were obtained by dividing the total number of cells
into the total number of patches.
Table 3. Occurrence of AChR patches on the lower and upper surface of muscle cells whose appearance in phase contrast
was normal or deteriorated after drug treatment
z
hrt
o
o
3-1
o
•
•
Z
>
m
H
H
Cd
o
o
o
O
as
4
Increased adhesiveness at AChR patches
65
tested at half the minimum concentration which caused muscle cells to round up.
For these experiments the drug was added to the culture medium of 1 to 4-dayold cultures and the cultures were then followed for up to 2 days. At the
concentrations tested (see Table 3) each of the drugs caused some of the muscle
cells to show signs of deterioration which became more prominent with time.
The deterioration was characterized by a loss of the smooth appearance of the
cell surface and a partial disruption of the cell striations (Fig. 5). At these
concentrations the drugs were also effective in abolishing, within 2 h, growthcone activity in cultures of Xenopus dorsal root ganglia and sympathetic
ganglia. Table 3 summarizes the frequency of occurrence of AChR patches in
the presence of the drugs. Those muscle cells which appeared normal when
viewed with phase contrast optics usually had AChR patches (Fig. 5), even after
2 days in colchicine, 2 days in cytochalasin B or 11 h in dibucaine. Furthermore
in cases where the number of AChR patches per cell was measured, the value
before and after treatment did not change markedly (Table 3). By contrast the
frequency of occurrence of AChR patches was reduced on those cells which had
a degenerated appearance, but even some of these cells still had well-defined
AChR patches (Table 3). Dimethylsulphoxide (1 %), which was used for
dissolving cytochalasin B (see Methods), did not by itself cause any cell degeneration or loss of AChR patches (Table 3). From these results it appears that
colchicine, cytochalasin B and dibucaine do not disrupt AChR patches independent of other cytotoxic effects.
DISCUSSION
The present study indicates that AChR patches on the lower surface of embryonic Xenopus muscle cells in culture are located at sites where the cells are more
strongly adherent to the culture dish. A similar conclusion has been reported for
AChR patches on the lower surface of rat myotubes in culture (Bloch &
Geiger, 1980). This increased adhesiveness at AChR patches bears a certain
resemblance to the situation at the adult neuromuscular junction where there is
a strong adhesion between the nerve terminal and the postsynaptic membrane
which contains a high density of AChRs (Betz & Sakmann, 1971, 1973). Our
finding thus extends the list of similarities between AChR patches on cultured
Xenopus muscle cells and the postsynaptic membrane at the neuromuscular
junction. In addition to being sites of high AChR density and of adhesion,
many of the AChR patches, like the postsynaptic membrane, are also sites of
localized cholinesterase (ChE) activity (Moody-Corbett & Cohen, 1981).
In turn the ChE is located at sites where the sarcolemma has basal lamina-like
material on its outer surface and fine filamentous material on its inner surface
(Weldon, Moody-Corbett & Cohen, 1981; see also Weldon & Cohen, 1979).
Similar ultrastructural specializations are found associated with the outer and
inner surface of the postsynaptic membrane at the neuromuscular junction
66
F. MOODY-CORBETT AND M. W. COHEN
(Ellisman et al. 1976; Fertuck & Salpeter, 1976; Kullberg, Lentz & Cohen,
1977; Heuser, 1980; Couteaux, 1981) and with AChR patches on embryonic
chick muscle cells in vivo and in culture (Jacob & Lentz, 1979; Burrage &
Lentz, 1981). The association of basal lamina material with the AChR patches
on cultured Xenopus muscle cells has also been established by employing
appropriate antibodies (Anderson & Fambrough, 1981) and may account for
the increased adhesiveness to the culture dish at these sites. This would be akin
to the situation at the neuromuscular junction where it has been established that
the basal lamina acts as an adhesive material between the nerve terminal and
the postsynaptic membrane (Betz & Sakmann, 1971, 1973).
The development of adhesive material at sites of high AChR density may have
important relevance for the development of the neuromuscular junction. The
most obvious possibility is that this material participates in the strong adhesion
that develops between the pre- and postsynaptic membranes. Furthermore, if
the adhesive material is indeed a component of the basal lamina at AChR
patches it might have other functional capabilities similar to those of the basal
lamina at the adult neuromuscular junction. For example, adult synaptic basal
lamina can induce a local differentiation of regenerating motor axons into
synaptic terminals (Sanes, Marshall & McMahan, 1978; McMahan et al.
1980 a, b) and it can also act as a focus for the accumulation of AChRs in the
sarcolemma (Burden et al. 1979; McMahan et al. 1980a, b). For the adhesive
material on embryonic muscle cells in culture to act as a focus for AChR
accumulation, it would, of course, have to appear on the cell surface before the
AChR patches. In the present study the increased adhesion at AChR patches
was already apparent in 2-day-old cultures (see Fig. 3) thereby indicating that
both properties develop in close temporal relationship. However our data do
not permit any conclusion concerning the sequence of development of the
increased adhesion and the AChR patches.
The occurrence of AChR patches at sites of strong adhesion with the culture
dish is consistent with recent studies which have found that AChR clusters on
rat myotubes in culture occur at sites of close contact with the culture dish
(Axelrod, 1980, 1981; Bloch & Geiger, 1980). In culture, such adhesion plaques
on other cell types occur at sites of the cell margin which are associated with the
internal support system involving filamentous material (Grinnell, 1978). It may
be that the sites where AChR patches and their associated specializations form
are determined by how the internal support system becomes organized during
cell spreading and growth. Such a mechanism could also apply to AChR
patches which form on the upper surface in the apparent absence of any contact
with a solid substrate (Fischbach & Cohen, 1973; Hartzell & Fambrough, 1974;
Anderson et al. 1977; Bloch & Geiger, 1980). In Xenopus muscle cells upper
surface AChR patches and their associated specializations tend to be located in
the central regions where the cells are thicker (Anderson et al. 1977; MoodyCorbett & Cohen, 1981; Weldon et al. 1981). An internal support system
Increased adhesiveness at AChR patches
67
involved in the development of such cell geometry might act as a focus for the
accumulation of AChRs and related specializations. However, our attempts in
the present study to disrupt AChR patches with drugs which are known to
disrupt cytoskeletal elements have not been successful. Cases where colchicine or
cytochalasin B reduced the incidence of AChR patches on muscle cells were
accompanied by cell degeneration. It is unclear, therefore, whether the drugs
acted by disrupting any of the specializations associated with AChR patches or
whether their action on AChR patches was secondary to other cytotoxic effects.
Other studies using colchicine, cytochalasin B and dibucaine have likewise
revealed little disruption of AChR accumulations (Axelrod et al 1978; Orida &
Poo, 1978; Bloch, 1979). It may be that the internal structural and molecular
specializations which are associated with sites of high AChR density (e.g.
Heuser & Salpeter, 1979; Bloch & Geiger, 1980; Cartaud et al 1981; Couteaux,
1981; Froehner et al 1981; Hall et al. 1981) do play a role in AChR patch
survival but are unaffected by these drugs. An alternative possibility which also
remains to be investigated is that the associated basal lamina can itself maintain
the survival of AChR patches.
This work was supported by the Medical Research Council of Canada. Personal support
to F.M.-C. from the Faculty of Medicine, McGill University and to M.W.C. from Le Conseil
de la Recherche en Sante du Quebec is also gratefully acknowledged. We thank Dr R. I.
Birks for the use of his microscope facilities and for continued interest in this work. Mr C.
Cantin provided expert technical assistance.
REFERENCES
ANDERSON, M. J. & COHEN, M. W. (1974). Fluorescent staining of acetylcholine receptors in
vertebrate skeletal muscle. J. Physiol., Lond. 237, 385-400.
ANDERSON, M. J. & COHEN, M. W. (1977). Nerve-induced and spontaneous redistribution of
acetylcholine receptors on cultured muscle cells. / . Physiol., Lond. 268, 757-773.
ANDERSON, M. J. & FAMBROUGH, D. M. (1981). Nerve-induced deposition of basal lamina
during the development of an amphibian neuromuscular junction in cell culture. Proc. Soc.
Neurosci. 7,670.
ANDERSON, M. J., COHEN, M. W. & ZORYCHTA, E. (1977). Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. /. Physiol, Lond. 268, 731-756.
AXELROD, D. (1980). Crosslinkage and visualization of acetylcholine receptors on myotubes
with biotinylated a-bungarotoxin and fluorescent avidin. Proc. natn. Acad. Sci., U.S.A. 77,
4823-4827.
AXELROD, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence./. CellBiol 89,141-145.
AXELROD, D., RAVDIN, P., KOPPEL, D. E., SCHLESSINGER, J., WEBB, W. W., ELSON, E. L. &
PODLESKI, T. R. (1976). Lateral motion offluorescentlylabelled acetylcholine receptors in
membranes of developing muscle cells. Proc. natn. Acad. Sci., U.S.A. 73, 4594-4598.
D., RAVDIN, P. M. & PODLESKI, T. R. (1978). Control of acetylcholine receptor
mobility and distribution in cultured muscle membranes. Biochim. biophys. Ada 511,23-38.
BEKOFF, A. & BETZ, W. H. (1976). Acetylcholine hotspots: development on myotubes
cultured from aneural limb buds. Science, N.Y. 193, 915-917.
BETZ, W. & SAKMANN, B. (1971). 'Disjunction' of frog neuromuscular synapses by treatment
with proteolytic enzymes. Nature, New Biol. Ihl, 94-95.
AXELROD,
68
F. MOODY-CORBETT AND M. W. COHEN
W. & SAKMANN, B. (1973). Effects of proteolytic enzymes on function and structure of
frog neuromuscular junction. /. Physiol., Lond. 230, 673-688.
BLOCH, R. (1979). Dispersal and reformation of acetylcholine receptor clusters of cultured
rat myotubes treated with inhibitors of energy metabolism. /. Cell Biol. 82, 626-643.
BLOCH, R. J. & GEIGER, B. (1980). Localization of acetylcholine receptor clusters in areas of
cell-substrate contact in cultures of rat myotubes. Cell 21, 25-35.
BURDEN, S. J., SARGENT, P. B. & MCMAHAN, U. J. (1979). Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of nerve. /. Cell
Biol. 82,412-425.
BURRAGE, T. G. & LENTZ, T. L. (1981). Ultrastructural characterization of surface specializations containing high-density acetylcholine receptors on embryonic chick myotubes in vivo
and in vitro. Devi Biol. 85,267-286.
CARTAUD, J., SOBEL, A., ROUSSELET, A., DEVAUX, P. F. & CHANGEUX, J. P. (1981). Consequences of alkaline treatment for the ultrastructure of the acetylcholine receptor-rich
membranes from Torpedo marmorata electric organ. /. Cell Biol. 90, 418-426.
COHEN, M. W. & WELDON, P. R. (1980). Localization of acetylcholine receptors and synaptic
ultrastructure at nerve-muscle contacts in culture: dependence on nerve type. /. Cell Biol.
86, 388-401.
COUTEAUX, R. (1981). Structure of the subsynaptic sarcoplasm in the interfolds of the frog
neuromuscular junction. /. Neurocytol. 10, 947-962.
EDELMAN, G. M. (1976). Surface modulation in cell recognition and cell growth. Science,
N.Y. 192,218-226.
ELLISMAN, M. H., RASH, J. F., STAEHELIN, A. & PORTER, K. R. (1976). Studies of excitable
membranes. II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers. /. Cell Biol. 68,
752-774.
FAMBROUGH, D. M. (1979). Control of acetylcholine receptors in skeletal muscle. Physiol.
Rev. 59,165-227.
FERTUCK, H. C. & SALPETER, M. M. (1976). Quantitation of junctional and extra-junctional
autoradiography after 125I-a-bungarotoxin binding at mouse neuromuscular junctions.
J.Cell Biol. 69,144-158.
FISCHBACH, G. D. & COHEN, S. A. (1973). The distribution of acetylcholine sensitivity over
uninnervated and innervated muscle fibers grown in cell culture. Devi Biol. 31, 147-162.
FRANK, F. & FISCHBACH, G. D. (1979). Early events in neuromuscular junction formation
in vitro. Induction of acetylcholine receptor clusters in the postsynaptic membrane and
morphology of newly formed synapses. /. Cell Biol. 83, 143-158.
BETZ,
FROEHNER, S. C , GULBRANDSEN, V., HYMAN, C , JENG, A. Y., NEUBIG, R. R. & COHEN, J. B.
(1981). Immunofluorescence localization at the mammalian neuromuscular junction of the
Mr 43000 protein of Torpedo post-synaptic membranes. Proc. natn, Acad. Sci., U.S.A. 78,
5230-5234.
FUKUDA, J., HENKART, M. P., FISCHBACH, G. D. & SMITH, T. G. (1976). Physiological and
structural properties of colchicine-treated skeletal muscle cells grown in tissue culture.
Devi Biol. 49,395-411.
GRINNELL, F. (1978). Cellular adhesiveness and extracellular substrata. Int. Rev. Cytol. 53,
65-144.
HALL, Z. W., LUBIT, B. W. & SCHWARTZ, J. H. (1981). Cytoplasmic actin in postsynaptic
structures at the neuromuscular junction. /. Cell Biol. 90, 789-792.
HARTZELL, H. C. & FAMBROUGH, D. M. (1973). Acetylcholine receptor production and
incorporation into membranes of developing muscle fibers. Devi Biol. 30, 153-165.
HEUSER, J. (1980). 3-D visualization of membrane and cytoplasm specializations at the frog
neuromuscular junction. In Ontogenesis and Functional Mechanisms of Peripheral Synapses
(ed. J. Taxi) 139-155. Amsterdam: Elsevier/North Holland.
HEUSER, J. F. & SALPETER, S. R. (1979). Organization of acetylcholine receptors in quickfrozen, deep-etched, rotary-replicated Torpedo postsynaptic membrane. /. Cell Biol. 82,
150-173.
Increased adhesiveness at AChR patches
69
M. & LENTZ, T. L. (1979). Localization of acetylcholine receptors by means of horseradish peroxidase-a-bungarotoxin during formation and development of the neuromuscular
junction in the chick embryo. / . Cell Biol. 82,195-211.
KULLBERG, R. W., LENTZ, T. L. & COHEN, M. W. (1977). Development of the myotomal
neuromuscular junction in Xenopus laevis: an electrophysiological and fine-structural
study. Devi Biol. 60,101-129.
LAND, R. R., PODLESKI, T. R., SALPETER, E. E. & SALPETER, M. M. (1977). Acetylcholine
receptor distribution on myotubes in culture correlated to acetylcholine sensitivity.
/. PhysioL, Lond. 269,155-176.
MCMAHAN, H. J., EDGINGTON, D. R. & KUFFLER, D. P. (1980a). Factors that influence
regeneration of the neuromuscular junction. / . exp. Biol. 89, 31-42.
MCMAHAN, H. J., SARGENT, P. B., RUBIN, L. L. & BURDEN, S. J. (19806). Factors that
influence the organization of acetylcholine receptors in regenerating muscle are associated
with the basal lamina at the neuromuscular junction. In Ontogenesis and Functional
Mechanisms of Peripheral Synapses (ed. J. Taxi) 345-354. Amsterdam: Elsevier/North
Holland.
MOODY-CORBETT, F. & COHEN, M. W. (1981). Localization of cholinesterase at sites of high
acetylcholine receptor density on embryonic amphibian muscle cells cultured without
nerve./. Neurosci. 1,596-605.
MOODY-CORBETT, F. & COHEN, M. W. (1982). Influence of nerve on the formation and
survival of acetylcholine receptor and cholinesterase patches on embryonic Xenopus
muscle cells in culture. / . Neurosci. 2, 633-646.
NICOLSON, G. L. & POSTE, G. (1976). Cell shape changes and transmembrane receptor
uncoupling induced by tertiary amine local anesthetics. J. Supramolec. Struct. 5, 65-72.
NICOLSON, G. L., SMITH, J. R. & POSTE, G. (1976). Effects of local anesthetics on cell morphology and membrane associated cytoskeletal organization in BALB/3T3 cells. / . Cell
Biol. 68, 395-402.
NIEUWKOOP, P. D. & FABER, J. (1967). Normal Table of Xenopus laevis (Daudin), 2nd ed.
Amsterdam: North Holland.
ORIDA, N. & Poo, M.-M. (1978). Electrophoretic movement and localization of acetylcholine
receptors in the embryonic cell membrane. Nature, Lond. 275, 31-35.
PENG, H. B., CHENG, P.-C, & LUTHER, P. W. (1981). Formation of AChR clusters induced by
positively charged latex beads. Nature, Lond. 292,831-834.
POSTE, G., PAPAHADJOPOULOUS, D., JACOBSON, K. & VAIL, W. J. (1975a). Effects of local
anesthetics on membrane properties. II. Enhancement of the susceptibility of mammalian
cells to agglutination by plant lectins. Biochim. biophys. Acta 394, 520-539.
POSTE, G., PAPAHADJOPOULOUS, D. & NICOLSON, G. L. (19756). Local anesthetics affect
transmembrane cytoskeletal control of mobility and distribution of cell surface receptors.
Proc. natn. Acad. Sci., U.S.A. 72,4430-4434.
POWELL, J. A. & FRIEDMAN, B. A. (1977). Electrical membrane activity: effect on distribution,
incorporation and degradation of acetylcholine receptors in the membranes of cultured
muscle. J. Cell Biol. 75, 321 a.
SANES, J. R., MARSHALL, L. M. & MCMAHAN, U. J. (1978). Reinnervation of muscle fiber
basal lamina after removal of myofibers. Differentiation of regenerating axons at original
synaptic sites./. Cell Biol. 78,176-198.
SYTKOWSKI, A. J., VOGEL, Z. & NIRENBERG, M. W. (1973). Development of acetylcholine
receptor clusters on cultured muscle cells. Proc. natn. Acad. Sci., U.S.A. 70, 270-274.
JACOB,
VOGEL, Z., SYTKOWSKI, A. J. & NIRENBERG, M. W. (1972). Acetylcholine receptors of muscle
grown in vitro. Proc. natn. Acad. Sci., U.S.A. 69, 3180-3184.
P. R. & COHEN, M. W. (1979). Development of synaptic ultrastructure at neuromuscular contacts in an amphibian cell culture system. / . Neurocytol. 8, 239-259.
WELDON, P. R., MOODY-CORBETT, F. & COHEN, M. W. (1981). Ultrastructure of sites of
cholinesterase activity on amphibian embryonic muscle cells cultured without nerve.
Devi Biol. 81, 341-350.
WELDON,
{Received 4 January 1982, revised 12 June 1982)