* Department of Physiology, McGill University, Montreal, Quebec, Canada H3G1Y6; Centre for Research in Neuroscience,
McGill University, Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4; § Department of
Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854; and
MRIC Biotechnology Group,
The North East Wales Institute, Plas Coch, Mold Road, Wrexam, United Kingdom LL11 2AW
The effect of laminin on the distribution of
dystroglycan (DG) and other surface proteins was examined by fluorescent staining in cultures of muscle
cells derived from Xenopus embryos. Western blotting
confirmed that previously characterized antibodies are reactive in Xenopus. In control cultures, DG,
DG,
and laminin binding sites were distributed as microclusters (<1 µm2 in area) over the entire dorsal surface of
the muscle cells. Treatment with laminin induced the
formation of macroclusters (1-20 µm2), accompanied
by a corresponding decline in the density of the microclusters. With 6 nM laminin, clustering was apparent
within 150 min and near maximal within 1 d. Laminin
was effective at 30 pM, the lowest concentration tested.
The laminin fragment E3, which competes with laminin
for binding to
DG, inhibited laminin-induced clustering but did not itself cluster DG, thereby indicating that other portions of the laminin molecule in addition to its
DG binding domain are required for its clustering activity. Laminin-induced clusters also contained dystrophin, but unlike agrin-induced clusters, they did not
contain acetylcholine receptors, utrophin, or phosphotyrosine, and their formation was not inhibited by a tyrosine kinase inhibitor. The results reinforce the notion
that unclustered DG is mobile on the surface of embryonic muscle cells and suggest that this mobile DG can
be trapped by at least two different sets of molecular interactions. Laminin self binding may be the basis for
the laminin-induced clustering.
That DG also plays a role in clustering AChRs at the
neuromuscular junction is based on the following evidence. In addition to being concentrated at the neuromuscular junction together with other DAPs and utrophin,
Since agrin is a ligand for Membrane Protein Isolation and Western Blot Analysis
For tests on Equal amounts (20 µg) of solubilized heavy microsomal membranes
from rabbit and Xenopus muscle were electrophoretically separated on
7.5% SDS-PAGE gels (Laemmli, 1970 For tests on dystrophin and utrophin, human and Xenopus tissues were
homogenized in 4 ml/g of extraction buffer (2% SDS, 5% 2-mercaptoethanol, 5% sucrose, 62.5 mM Tris-HCl, pH 6.8) using a Polytron homogenizer as previously described (Nguyen thi Man et al., 1990). The homogenates were boiled for 2 min and clarified by centrifugation at 100,000 g for
20 min. The extracts were subjected to SDS-PAGE on 3-12.5% gradient
gels, together with prestained molecular mass markers, and electrophoretically transferred to nitrocellulose membranes for 16 h at 100 mA in 25 mM
Tris, 192 mM glycine, 0.003% SDS. The Western blots were developed
with 1:100 dilutions of mAb culture supernatants followed by a biotinylated anti-mouse Ig and avidin-peroxidase detection system (Vectastain; Vector Labs Inc., Peterborough, UK) as previously described (Nguyen thi
Man et al., 1991).
Cultures
Myotomal muscle cells derived from 1-d-old Xenopus embryos were
plated in glass culture chambers as described previously (Cohen et al.,
1994 Fluorescent Staining
For extracellular epitopes, cultures were stained alive at 6-8°C and then
fixed. To stain intracellular epitopes, it was necessary to fix and permeabilize the cells (see Fig. 5, A and B) either with precooled (
AChRs were stained with rhodamine-conjugated The widespread distribution of Analysis
Because of their large size, laminin and mAb IIH6 (an IgM) have little if
any access to the ventral muscle cell surface which is in contact with the
culture substrate. Accordingly, LBS and Negatives on 35-mm film (Kodak T-MAX 3200) were analyzed using
the MCID imaging system (Imaging Research Inc., St. Catharines, ON,
Canada) at a magnification of 208 pixels/µm2. For each frame, a rectangular region of interest (ROI) was chosen where all the clusters were in focus. ROIs were typically 200-400 µm2. The area of each discrete cluster
within the ROI was recorded, thereby revealing the size distribution of
the clusters, which were arbitrarily divided into microclusters (<1 µm2 in
area) and macroclusters (1-20 µm2 in area). The percentage of the ROI's
area occupied by macroclusters was used as a measure of laminin-induced
clustering. Another measure was the decline in the density of microclusters. This density was calculated as the number of microclusters within the
ROI divided by the difference between the ROI area and the total macrocluster area.
Since microclusters and laminin-induced macroclusters were both readily
resolved after staining for LBS, photographs of this immunofluorescence
were used for most of the quantitative analysis. One problem, however,
was that the intensity of the immunofluorescence of the laminin-induced
macroclusters was considerably greater than that of the microclusters. Accordingly, photographic exposure times that were optimal for the microclusters resulted in overexposure and some "blooming" of the macroclusters. Conversely, shorter photographic exposure times which were optimal
for the macroclusters resulted in underexposure and some apparent loss
of the microclusters. To avoid exaggerating the laminin-induced decline in
the density of microclusters we opted for the longer exposure times (5-10 s).
Subsequently, we adjusted the intensity of the light table on which the
negative was placed to exclude from the measurements as much of the
"blooming" as possible without the loss of microclusters.
In cultures stained with two different probes, colocalization was assessed on pairs of photographs printed at a magnification of 1,600×. A
transparency was placed over one of the photographs, and the position of
the fluorescein (or rhodamine) fluorescence was marked on the transparency. The transparency was then repositioned over the companion photograph, thereby revealing the location of the fluorescein fluorescence with
respect to the rhodamine fluorescence.
Counts of cells with AChR-containing megaclusters were made directly
on the microscope using an occular graticule to assess cluster size. Clusters
whose diameters were at least 5 µm were categorized as megaclusters.
Reactivity of Anti- Based on previous Western blot analysis, the anti-
Fig. 2 shows tests for the anti-dystrophin mAb MANEX
7374-10A11 and for the anti-utrophin mAb MANCHO 3. In human tissues, dystrophin is present in muscle but undetectable in lung (Fig. 2 B). By contrast, utrophin is
present in most human tissues, including lung (Fig. 2 A),
but is barely detectable in normal human muscle where it
is confined to neuromuscular junctions, blood vessels, and
nerve fibers (Nguyen thi Man et al., 1991). The anti-utrophin mAb also appears to show a high degree of specificity for Xenopus utrophin; it gives a strong band in the heart
but not in skeletal muscle (Fig. 2 A), although dystrophin
is abundant in Xenopus skeletal muscle (Fig. 2 B). Conversely, the anti-dystrophin mAb clearly recognizes Xenopus dystrophin in skeletal muscle, but does not cross react
with the abundant utrophin in Xenopus heart (Fig. 2 B).
We therefore used these mAbs for immunofluorescent staining of embryonic Xenopus muscle cell cultures.
Clusters in Control Cultures
Based on fluorescent staining for Megaclusters are >20 µm2 in area and often larger than
40 µm2. When viewed at high magnification some of them
appear uniform (see Fig. 10 D), but most have a complex,
patterned appearance (Fig. 3, B and C). Megaclusters contain similar distributions of
Macroclusters, defined as 1-20 µm2 in area, are usually
1-3 µm2 and rarely as large as 10 µm2. They typically occur near the ends of some of the muscle cells in control
cultures and can be seen after staining for Microclusters, defined as discrete sites of immunofluorescence having an area of <1 µm2, are distributed in a
random fashion over the entire dorsal surface of the muscle cells. They were readily observed after staining for The density of microclusters stained for As shown in Fig. 4, A and B, the size distributions of the
clusters stained for Laminin-induced Clustering
When cultures were treated with laminin at room temperature, macroclusters of a variety of shapes and sizes formed
over the entire dorsal surface of the muscle cells. Invariably, the regions surrounding these laminin-induced macroclusters had a reduced density of microclusters (Fig. 5 A).
As shown in Fig. 4 C, the size distribution of the clusters
in laminin-treated cultures formed a continuum, with no
demarcation between microclusters and macroclusters.
Rather, the laminin treatment reduced the percentage of
smallest microclusters and increased the percentage of
macroclusters. In the example of Fig. 4 C, the microclusters outnumbered the macroclusters by about 10-fold, but
because of size differences, the macroclusters actually occupied a greater proportion of the surface area.
The laminin-induced macroclusters appeared to be randomly distributed, were readily resolved with LBS and
The time and concentration dependence of the laminininduced changes are summarized in Fig. 6 A, which shows
the percentage of surface area occupied by the LBS macroclusters, and Fig. 6 B, which shows the density of the
LBS microclusters. In control cultures which were exposed
to 0.6-6 nM laminin for 10-150 min at 6-8°C (Fig. 6, open
columns), the macroclusters occupied only 0.2-1.2% of the
surface area, and the density of the microclusters varied
from 1.34-1.77/µm2. When cultures were exposed to 6 nM
laminin at 22-24°C (Fig. 6, shaded columns) an increase in
macrocluster formation was apparent within 150 min, and
this was accompanied by a decrease in the density of the
microclusters. Larger, temperature-dependent changes occurred with more prolonged treatment. After 1 d in 6 nM
laminin the macroclusters occupied 10.2% of the surface
area, and the density of microclusters was only 0.26/µm2.
Significant laminin-induced clustering occurred within 1 d
even at a concentration of 0.03 nM, the lowest concentration tested. Intermediate concentrations of laminin induced
intermediate amounts of clustering. Extending the laminin
treatment beyond 1 d resulted in some additional clustering, especially at the lower concentrations of laminin.
Taken together the findings indicate that low concentrations of laminin bind to Effects of the Laminin Fragment E3
E3, one of the fragments obtained when laminin is cleaved
by elastase, consists of the last two G repeats at the end of
the long arm of the laminin molecule. In agreement with
previous findings that this portion of the molecule binds to
In view of its lack of clustering activity and its known
competition with laminin for binding to Overall, the results indicate that laminin-induced clustering depends not only on that portion of the laminin
molecule which binds to Comparison with Agrin-induced Clustering
Previous studies on other culture systems have indicated
that agrin-induced clusters contain DG and several other
molecules including tyrosine phosphorylated AChRs, other
tyrosine phosphorylated proteins, and utrophin (Fallon
and Hall, 1994
In contrast to agrin-induced clusters, the vast majority of
laminin-induced clusters did not contain detectable concentrations of AChRs, utrophin, or PY (Fig. 10, B-D).
However, the laminin-induced clusters did contain dystrophin (Fig. 10, A and B). The effect of the tyrphostin
RG50864, a tyrosine kinase inhibitor (Lyall et al., 1989 Table I.
Inhibition of Agrin-induced Clustering by a
Tyrphostin
DYSTROGLYCAN (
DG)1 and
DG are present in a variety of tissues including brain but have been studied most
intensively in skeletal muscle, where they are thought to
play a role in maintaining the structural integrity of the
sarcolemma and in clustering acetylcholine receptors
(AChRs) at the neuromuscular junction (Fallon and Hall, 1994
; Tinsley et al., 1994; Campbell, 1995
; Carbonetto and
Lindenbaum, 1995
; Ozawa et al., 1995
). These dystrophin
associated proteins (DAPs) exist as a complex, bound
tightly to each other, and are derived from the same precursor protein (Ibraghimov-Beskrovnaya et al., 1992
,
1993; Bowe et al., 1994
; Yoshida et al., 1994
).
DG is a
highly glycosylated extracellular peripheral membrane protein that binds laminin and agrin and can thereby establish a link with the extracellular matrix in extrajunctional regions as well as the neuromuscular junction (IbraghimovBeskrovnaya et al., 1992; Ervasti and Campbell, 1993
; Gee
et al., 1993
, 1994; Campanelli et al., 1994
). Its glycosylated
transmembrane partner
DG can bind dystrophin (Suzuki
et al., 1994
; Jung et al., 1995
). Since dystrophin can interact
with cytoskeletal actin (Hemmings et al., 1992
), this complex of molecules is thought to provide a link from the extracellular matrix to the cytoskeleton. The dystrophinrelated protein utrophin substitutes for dystrophin at sites
where AChRs are clustered (Fallon and Hall, 1994
; Tinsley et al., 1994; Carbonetto and Lindenbaum, 1995
) and is
also complexed with DG in nonmuscle cells which lack
dystrophin (Matsumura et al., 1993
, James et al., 1996
).
Other transmembrane DAPs such as the muscle-specific
sarcoglycan complex as well as intracellular peripheral
membrane DAPs such as the syntrophins are also associated with this extracellular matrix-cytoskeleton link (Kramarcy et al., 1994
; Suzuki et al., 1994
). The notion that this
complex linkage is an important structural element is supported by the association of different forms of muscular
dystrophy with genetic mutations in dystrophin, the sarcoglycans, and the laminin
2 chain (Campbell, 1995
; Ozawa
et al., 1995
; Worton, 1995
).
DG is the major agrin-binding protein on the surface of
skeletal muscle cells (Bowe et al., 1994
; Campanelli et al.,
1994
; Gee et al., 1994
). Agrin is deposited by growing nerve
fibers at newly forming synapses on muscle cells (Cohen and Godfrey, 1992
) and is the major neural agent involved
in triggering the aggregation of AChRs at these sites in
culture (Reist et al., 1992
) as well as in vivo (Gautam et al.,
1996
).
DG accumulates very early together with AChRs
and neurally released agrin at newly forming synapses,
within as little as 1 h after the establishment of nerve-muscle contact (Cohen et al., 1995a
). Clusters of AChRs induced by the addition of agrin to the culture medium of
embryonic muscle cells likewise contain an accumulation
of
DG as well as other DAPs and utrophin (Campanelli
et al., 1994
; Gee et al., 1994
). Tyrosine phosphorylation
has been strongly implicated in agrin's clustering activity
(Wallace, 1994
, 1995
; Ferns et al., 1996
), and recent data
indicate that this phosphorylation depends upon activation of the transmembrane receptor tyrosine kinase MuSK
by agrin (DeChiara et al., 1996
; Glass et al., 1996
). Glass et al. suggest that
DG could play a role in this activation
by helping to present agrin to MuSK.
DG and can cluster it, it
seemed of interest to explore whether laminin, the more
ubiquitous ligand, would also cluster DG. In fact, laminininduced changes in the distribution of its binding sites
were briefly reported in a study on cultured rat myotubes
(Vogel et al., 1983
). In the present study we have examined this action of laminin in more detail in cultures of embryonic Xenopus muscle cells. We report that DG is clustered by laminin, that these clusters contain dystrophin, and that this laminin-induced clustering involves a corresponding depletion of DG in regions surrounding the clusters. The laminin fragment E3 has no clustering activity
but inhibits laminin's. Unlike nerve and agrin-induced
clustering, laminin-induced clustering of DG is not accompanied by an accumulation of AChRs or utrophin and occurs in the absence of tyrosine phosphorylation. The findings support the notion that unclustered DG is mobile on
the surface of embryonic muscle cells and suggest that this
mobile DG can be trapped by at least two different sets of
molecular interactions. Since laminin, a t-shaped molecule,
binds to
DG through its G domain at the end of its long
arm (Ibraghimov-Beskrovnaya et al., 1992
; Ervasti and
Campbell, 1993
; Gee et al., 1993
) and can cross link with
itself through the terminal regions of its short arms (Yurchenco and Cheng, 1993
; Colognato-Pyke et al., 1995
), we
speculate that this self binding property of laminin causes
mobile DG to cluster.
Materials and Methods
DG, membrane proteins were isolated from leg muscle of
the frog Xenopus laevis and from leg and back muscles of rabbit as described previously (Cohen et al., 1995a
). Briefly, muscle was homogenized
in a Polytron mixer (Brinkmann Instruments Inc., Rexdale, ON, Canada)
in 7.5 vol of homogenization buffer (20 mM sodium pyrophosphate, 20 mM
sodium phosphate monohydrate, 1 mM MgCl2, 0.303 M sucrose, 0.5 mM
EDTA, pH 7.0) in the presence of the following protease inhibitors: aprotinin (1 µM), leupeptin (1 µM), pepstatin A (1 µM), benzamidine (1 mM),
iodoacetamide (1 mM), and PMSF (1 mM). The homogenate was centrifuged at 14,000 g at 4°C for 15 min. The supernatant was retained and the
pellet re-extracted (and recentrifuged) in 75% of the original buffer volume. The ensuing supernatants were pooled and centrifuged at 30,000 g
for 30 min at 4°C to pellet the heavy microsome fraction. The pellet was
then resuspended in 0.6 M KCl, 0.303 M sucrose, 50 mM Tris-HCl, pH 7.4, with protease inhibitors and incubated on ice. After 30 min this suspension was centrifuged at 142,000 g for 30 min at 4°C. The KCl-washed
heavy microsomes (pellet) were solubilized on ice for 30 min in 1% digitonin, 50 mM Tris-HCl, pH 7.4, with protease inhibitors. This material was
subjected to centrifugation at 85,000 g for 30 min at 4°C and the supernatant retained.
) and the protein subsequently transferred to nitrocellulose membranes. The blots were blocked in 10 mM
Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% Tween-20 with 5% skim milk. The
anti-
DG mAb 43DAG1/8D5 (Novocastra Laboratories Ltd., Newcastle
upon Tyne, UK) was equilibrated with the membrane in blocking buffer
for 30 min at room temperature. The blot was washed with repeated
buffer changes for 1 hour in the above buffer without skim milk and then
incubated with secondary antibody conjugated to horseradish peroxidase
(Sigma Chemical Co., St. Louis, MO). Excess secondary antibody was removed by washing for 2 h. Bound antibody was visualized using chemiluminescence (DuPont-New England Nuclear, Boston, MA).
). The coverslip which formed the floor of the chamber was coated
with rat tail collagen (Upstate Biotechnology, Inc., Lake Placid, NY) and
bovine plasma fibronectin (Sigma Chemical Co.). The culture medium
consisted of 67% (vol/vol) L15 and 0.08% (wt/vol) bovine albumin (fraction V; GIBCO BRL, Burlington, ON, Canada). Treatment with mouse
laminin-1, isolated from the EHS sarcoma (GIBCO BRL), with the E3
fragment of laminin (prepared as described by Yurchenco and Cheng,
1993
), or with an extract of Torpedo agrin (a generous gift from E.W. Godfrey, Medical College of Wisconsin, Milwaukee, WI) was begun after the
cells had been in culture for 1-3 d. The agrin extract was used at a dilution of 1:100-1:200. In cases in which the tyrphostin RG50864 (Rhône-Pulenc Rover Pharmaceuticals, Inc., Collegeville, PA) was included with laminin
or agrin, it was added to the culture up to 4 h earlier. Unless indicated otherwise the cultures were maintained at room temperature (22-24°C).
16°C) 95% ethanol for 5-10 min or with 4% (wt/vol) formaldehyde for 10-15 min, followed by 1% (vol/vol) Triton X-100 for 20 min. After fixation, all steps of
the staining protocol were carried out at 6-8°C. Permeabilized cultures
were exposed to 10% goat serum (in 67% L15) for 10 min before staining
them. Cultures were exposed to primary antibodies and fluorescent reagents for 30-60 min and rinsed with 1% goat serum. Stained cultures were stored in 4% formaldehyde in the refrigerator and subsequently processed for fluorescence microscopy (Cohen and Godfrey, 1992
).
Fig. 5.
Laminin-induced clusters of DG and LBS. (A1 and
A2)
DG and LBS immunofluorescence after treatment with 2.4 nM laminin for 3 d, revealing large numbers of macroclusters and
a greatly reduced density of microclusters (compare with Fig. 3 B).
(B1 and B2)
DG immunofluorescence in the absence of permeabilization and LBS immunofluorescence after the same laminin
treatment as in A. Only some slight "bleedthrough" of the bright
LBS immunofluorescence is seen in B1, thereby confirming that
the immunofluorescence in A1 involved the binding of the anti
DG mAb to an intracellular epitope. (C)
DG immunofluorescence on the ventral surface of a cell after the same laminin treatment as in A. (D)
DG immunofluorescence on the ventral
surface of a cell in a culture which was not treated with laminin.
Comparison of C and D reveals that the laminin treatment induced an extensive accumulation of
DG at the cell periphery but did not induce clustering over the rest of the ventral surface which is inaccessible to laminin. The laminin-induced clustering at the cell periphery is out of focus in A and B2. Bar, 5 µm.
[View Larger Version of this Image (155K GIF file)]
Fig. 3.
Distribution of DG and LBS on the dorsal surface of
muscle cells in control cultures. (A) DG immunofluorescence,
revealing microclusters and a few macroclusters. (B1)
DG immunofluorescence. Part of a megacluster is seen. Microclusters
are also apparent but are relatively faint. (B2) LBS immunofluorescence in same field as B1. Microclusters, as well as the megacluster, are well resolved. (C) Laminin immunofluorescence in the
absence of pretreatment with laminin, revealing a megacluster but
very few microclusters. (D1 and D2)
DG and LBS immunofluorescence, obtained by exposing the culture to 6 nM laminin for 10 min at low temperature, followed by staining for
DG and laminin. The brief pretreatment with laminin inhibited the staining
for
DG (compare D1 with A). Bar, 5 µm.
[View Larger Version of this Image (124K GIF file)]
-bungarotoxin (Molecular Probes, Inc., Eugene, OR) at 2-4 µg/ml. The following primary antibodies were used: 1:100 mAb IIH6 directed against
DG (Ervasti and Campbell, 1993
; Cohen et al., 1995a
); 1:500 anti-laminin, a polyclonal antibody
(GIBCO BRL) which also binds to the laminin fragment E3; 1:20-1:40
anti-
DG mAb 43DAG1/8D5; 5 µg/ml mAb 4G10, directed against phosphotyrosine (Upstate Biotechnology, Inc.); and anti-utrophin (MANCHO 3;
Nguyen thi Man et al., 1991) and anti-dystrophin mAbs (MANEX 737410A11; Morris et al., 1995
) at 1:5-1:20. Appropriate affinity purified secondary antibodies conjugated with fluorescein or rhodamine (Organon
Teknika, Inc., Scarborough, ON, Canada; Molecular Probes, Inc.) were
used at 10 µg/ml. For some experiments primary antibodies were followed
by biotin-conjugated secondary antibodies and fluorescent streptavidins
(Molecular Probes, Inc.). To stain for laminin binding sites (LBS) without inducing clustering, cultures were cooled to 6-8°C and then exposed to
0.6-6 nM laminin for 10-30 min, followed by anti-laminin and secondary
antibody. The latter protocol was also used when muscle cells were cultured for a day or more in the presence of a lower concentration of laminin.
DG microclusters seen in control cultures of the present study was not observed consistently in a previous
study (Cohen et al., 1995a
). Methodological differences probably account
for this discrepancy. In the latter study some of the cultures were stained
with a more dilute solution of mAb IIH6, and some were fixed before
staining. In addition, the culture substrate contained laminin rather than
fibronectin. Laminin competes with mAb IIH6 for binding to
DG (Ervasti and Campbell, 1993
) and reduces the intensity of mAb IIH6 immunofluorescence (see Fig. 3 D).
DG immunofluorescence as
well as laminin-induced clustering were restricted to the dorsal surface.
For photography and subsequent analysis, it was therefore necessary to
locate regions of the dorsal surface which were in a single focal plane. All
cultures were examined and photographed using a 100× oil immersion
objective.
Results
DG, -Dystrophin, and -Utrophin
Antibodies in Xenopus
DG
mAb IIH6 recognized Xenopus
DG (Cohen et al.,
1995a
). The results of similar tests made for the anti-
DG
mAb 43DAG1/8D5 are shown in Fig. 1. This mAb recognizes a single protein band on Western blots of adult rabbit and Xenopus skeletal muscle membrane extracts. In
comparison to rabbit
DG, the Xenopus protein migrates
a little more slowly on SDS-PAGE. This small difference
was also noted previously in tests with another anti-DG
antibody (Cohen et al., 1995a
).
Fig. 1.
Western blot analysis of rabbit and Xenopus
DG. Nitrocellulose transfers of heavy microsomes
from rabbit (R) and Xenopus (frog, F) muscle, which had
been separated by SDSPAGE, were stained with
mAb 43DAG1/8D5 raised
to 15 of the last 16 amino acids at the extreme COOH
terminus of the human dystroglycan sequence. It recognizes a band of roughly 43 kD in rabbit corresponding to the size of
DG (Ibraghimov-Beskrovnaya et al., 1992
).
A similar though somewhat
larger band of 44-46 kD is
visible in Xenopus. Molecular mass markers (in kD) are
shown to the right.
[View Larger Version of this Image (29K GIF file)]
Fig. 2.
Western blot analysis of human and Xenopus utrophin
(A) and dystrophin (B). The Xenopus (frog, F) tissues analyzed
were skeletal muscle (m), liver (lr), and heart (h), and the human
(H) tissues were skeletal muscle and lung (lg). Positions of molecular mass markers at 180, 116, and 84 kD are shown, while utrophin and dystrophin migrate at ~400 kD. Lower molecular mass
bands at ~50 and 120 kD are nonspecific and due to cross reactions of the second antibody system.
[View Larger Version of this Image (54K GIF file)]
DG,
DG, LBS, and
AChRs, clusters on the dorsal surface of embryonic Xenopus myotomal muscle cells in culture can be classified into
three size categories termed here megaclusters, macroclusters, and microclusters.
DG,
DG, LBS, and AChRs
although at their periphery,
DG,
DG, and LBS can extend beyond the limits of the AChRs (see Fig. 3 A in Cohen et al., 1995a
). The overall incidence of megaclusters on the dorsal surface is low. Many cells have none and usually have instead, one or two megaclusters on their ventral
surface (see Fig. 5, C and D and Fig. 11, A and C). The remaining cells rarely have more than one megacluster on
their dorsal surface. Megaclusters were not included in the
quantitative analysis shown in Figs. 4, 6, and 7.
Fig. 10.
Laminin-induced clusters contain dystrophin but lack
AChRs, utrophin, and PY. (A1 and A2) LBS and dystrophin immunofluorescence, revealing excellent colocalization. (B1 and B2)
Dystrophin immunofluorescence and AChR stain, revealing
AChRs at a megacluster but not at laminin-induced macroclusters. (C1 and C2) LBS and utrophin immunofluorescence. Utrophin was not detected at laminin-induced clusters (but was always
observed wherever AChRs were clustered). (D1 and D2) LBS and PY immunofluorescence, revealing PY at a megacluster but
not at the laminin-induced macroclusters. The laminin treatment
was 2.4 nM for 3 d in A and C, 6 nM for 2 d in B, and 6 nM for 1 d in D. Some slight bleedthrough of the bright LBS immunofluorescence is seen in C2 and D2. Bar, 5 µm.
[View Larger Version of this Image (111K GIF file)]
Fig. 11.
Effect of agrin
and laminin on megaclusters. Cultures were stained
for AChRs and photographed at low magnification. (A) In control cultures
each muscle cell typically
has one or two megaclusters. (B) After treatment with
agrin for 1 d, the muscle cells
have many agrin-induced
macroclusters but often lack
megaclusters. (C) After
treatment with 6 nM laminin
for 1 d, each muscle cell still
has one or two megaclusters.
Variable numbers of autofluorescent yolk granules are
apparent in the central regions of the cells. The faint
outlining of some of the cells
in C is due to bleedthrough of the very bright LBS immunofluorescence (not shown)
at cell peripheries. Bar, 25 µm.
[View Larger Version of this Image (83K GIF file)]
Fig. 4.
Sizes of individual DG and LBS clusters. (A)
DG
clusters (n = 4,989) in control cultures not exposed to laminin.
(B) LBS clusters (n = 7,930) in control cultures exposed briefly
(10-30 min) to laminin (0.6-6 nM) at low temperature immediately before staining. (C) LBS clusters (n = 3,621) after treatment with 6 mM laminin for 1 d at room temperature. In control
cultures (A and B) there were very few macroclusters (>1 µm2),
and most of the microclusters were <0.1 µm2. After treatment
with laminin for 1 d (C) the incidence of macroclusters was increased whereas the incidence of the smallest microclusters was
decreased.
[View Larger Version of this Image (8K GIF file)]
Fig. 6.
Effect of laminin
treatment on (A) the percentage of surface area occupied by LBS macroclusters
and (B) the density of LBS microclusters. Laminin concentrations (in nM) and exposure times are indicated at
the bottom of B. The cultures
were treated with laminin either at low temperature
(open columns) or at room
temperature (shaded columns). In some cases the
laminin treatment was carried out in the presence of
RG50864 (striped columns).
On average the means and
standard errors are based on
26 cells (range, 9-79).
[View Larger Version of this Image (37K GIF file)]
Fig. 7.
Relationship between the density of microclusters and
the percentage of surface area occupied by macroclusters. From
the data of Fig. 6.
[View Larger Version of this Image (13K GIF file)]
DG,
DG, or
LBS. However, unlike megaclusters, the great majority of
macroclusters do not contain AChRs.
DG
(Fig. 3 A). Immunofluorescent staining for
DG, known from biochemical studies to be bound tightly to
DG (Yoshida et al., 1994
), also revealed the microclusters (Fig. 3
B1) but not as consistently and with less resolution, presumably because of technical limitations such as reduced
immunoreactivity associated with fixation and permeabilization (see Materials and Methods). A widespread distribution of microclusters was also seen after staining for
LBS (Fig. 3 B2). Virtually all microclusters that were resolved with
DG immunofluorescence also contained
LBS (Fig. 3 B). Such colocalization is in line with biochemical studies showing that laminin binds to
DG which is
tightly complexed to
DG (Ibraghimov-Beskrovnaya et
al., 1992
; Ervasti and Campbell, 1993
; Gee et al., 1993
). In
the absence of pretreatment with exogenous laminin, laminin immunofluorescence revealed megaclusters but very
few microclusters (Fig. 3 C). A lack of endogenous laminin immunofluorescence over most of the cell surface, except
at AChR-containing clusters, was also reported for cultures of fetal rat muscle cells (Vogel et al., 1983
).
DG was 1.33 ± 0.09/µm2 (mean ± SEM; n = 17) whereas the density of
the LBS microclusters was 1.58 ± 0.04/µm2 (n = 95). Since
the latter value is only slightly greater than that obtained
for
DG microclusters and since laminin is known to bind
to
DG, it follows that most if not all of the LBS microclusters contained
DG. The slight difference between the
two densities may be related to the fact that the LBS immunofluorescence was generally brighter than the
DG
immunofluorescence. Furthermore, in agreement with previous findings that laminin competes with mAb IIH6 for
binding to biochemically isolated
DG (Ervasti and Campbell, 1993
), laminin reduced the
DG immunofluorescence in a dose-dependent manner and even a 10 min
pretreatment with 6 nM blocked much of the microcluster
type of
DG immunofluorescence (Fig. 3 D). Overall then,
the findings complement previous biochemical data and
suggest that
DG functions as a laminin receptor in situ.
DG and LBS were essentially the same,
with the microclusters greatly outnumbering the macroclusters. Interestingly, most of the microclusters were <0.1
µm2. This area corresponds to a diameter of <0.4 µm and
encroaches on the limit of resolution of the light microscope. Thus if the immunofluorescence emanating from a
single
DG molecule were bright enough to be photographed, its size would be similar to that of the smallest
microclusters photographed in this study. Alternatively, the smallest microclusters may have contained several
DG molecules.
DG immunofluorescence, but were considerably fainter
with
DG immunofluorescence, further reflecting the
competition noted between laminin and mAb IIH6 for
binding to
DG. A pronounced laminin-induced clustering was also apparent at cell peripheries where the dorsal
and ventral surfaces meet (Fig. 5 C). Combined staining
for LBS and
DG revealed that laminin was bound at all
sites of laminin-induced clustering of DG (Fig. 5 A). On
the other hand the laminin treatment produced no apparent change in the pattern of
DG immunofluorescence on the ventral surface which is inaccessible to laminin (Fig. 5, C and D), thus indicating that the laminin-induced clustering was restricted only to those sites where laminin binding occurred.
DG and induce an apparently
random clustering of it and its transmembrane partner
DG. This clustering is accompanied by a depletion of DG
in the surrounding regions. The decline in the density of
microclusters that accompanied the formation of macroclusters is shown in more detail in Fig. 7. The inverse relationship suggests that the macroclusters were derived from
the microclusters and can be readily explained by a clustering mechanism whereby the binding of laminin to mobile DG promotes aggregation of colliding laminin-DG
complexes (see Discussion). That the decline in microcluster density was most-pronounced at the onset of macrocluster formation is also readily explained by this mechanism. As pointed out earlier, the immunofluorescence
emitted from each of the smallest microclusters might be
due to a much smaller source (and perhaps even a single laminin-DG complex). If pairs of these microclusters aggregated, their density would be reduced by half, but the
size of the immunofluorescence associated with these
newly formed pairs might not be changed at all. Nor would
the area of the macroclusters be changed. In addition,
since the smallest microclusters were the most numerous (Fig. 4), these would require considerable recruitment of
surrounding mobile laminin-DG complexes to attain macrocluster dimensions. Based on these considerations, it is
not unexpected that the decline in microcluster density
was steepest at the onset of the laminin-induced clustering.
DG (Gee et al., 1993
), acute exposure of cultures to E3,
like acute exposure to laminin, resulted in widespread binding in the form of microclusters and inhibited the widespread binding of mAbIIH6. The latter inhibition was
41% in the presence of 33 nM E3 and virtually complete
(94%) in the presence of 3.3 µM E3. Unlike laminin, however, E3 did not exhibit any clustering activity. When cultures were treated for up to 2 d with 3.3 nM-3.3 µM E3 there
was no detectable clustering as assessed by staining for E3
binding sites (Fig. 8 A) or for
DG. The density of microclusters after exposure to 3.3 µM E3 for 2 d was 1.326 ± 0.105/µm2, and macroclusters occupied only 0.2 ± 0.1%
of the surface area (n = 13). Essentially similar values
(1.271 ± 0.053/µm2 and 0.5 ± 0.2%, respectively; n = 15)
were obtained for acute (30 min) exposure to 3.3 µM E3.
Fig. 8.
E3 lacks clustering activity and inhibits laminin-induced
clustering. (A) E3 binding sites after treatment with 3.3 µM E3 for
2 d. Note the relatively high density of microclusters and the paucity of macroclusters. (B) LBS after treatment with 0.6 nM laminin for 1 d. (C) Laminin and E3 binding sites after treatment with
0.6 nM laminin and 3.3 µM E3 for 1 d. E3 inhibited the laminininduced clustering. Bar, 5 µm.
[View Larger Version of this Image (167K GIF file)]
DG, E3 would
be expected to inhibit laminin-induced clustering. This was
found to be the case. On muscle cells treated with 0.6 nM
laminin for 1 d (Fig. 8 B), macroclusters occupied 4.9 ± 0.7% (n = 36) of the surface area, whereas in comparison,
cultures treated with a combination of 0.6 nM laminin and
3.3 µM E3 for 1 d (Fig. 8 C), macroclusters occupied only
0.9 ± 0.3 (n = 28) of the surface area. Thus E3 inhibited
formation of macroclusters by >80%. Lower concentrations of E3 also inhibited the laminin-induced clustering,
but to a lesser extent.
DG but also on other portions
which are not contained in the E3 fragment. The simplest
proposal is that these other portions are the self-binding
sites on the laminin molecule (see Discussion).
; Carbonetto and Lindenbaum, 1995
; Glass
et al., 1996
). As shown in Fig. 9, we confirmed that agrininduced clusters on embryonic Xenopus muscle cells contain DG, AChRs, utrophin, and phosphotyrosine (PY),
as well as LBS. These clusters spanned the macrocluster
range in size and also varied considerably in shape and distribution. Unlike the laminin-induced clusters, they were
not restricted to the dorsal surface and actually occurred
more frequently on the ventral surface. In regions of the
dorsal surface lacking agrin-induced clusters, the density of
the widespread LBS microclusters was almost unchanged
(1.43 ± 0.07/µm2 for agrin-treated cultures compared with
1.58 ± 0.04/µm2 for control cultures). However, decreases
in the density of microclusters were apparent in regions of
the dorsal surface where the incidence of agrin-induced
clusters was relatively high (Fig. 9 A2).
Fig. 9.
Agrin-induced clusters on the dorsal surface. (A1 and
A2) DG and LBS immunofluorescence, revealing several patterned clusters, excellent colocalization, and some decline in the
density of microclusters in surrounding regions. (B1 and B2)
AChR stain and utrophin immunofluorescence, revealing excellent colocalization. (C1 and C2) AChR stain and PY immunofluorescence, revealing excellent colocalization. The agrin treatment
was 3 d in A and B and 2 d in C. Similar results were obtained
when the agrin treatment was 1 d. Bar, 5 µm.
[View Larger Version of this Image (153K GIF file)]
),
was also different in the two cases. As shown in Table I,
100 µM RG50864 markedly inhibited the agrin-induced clustering (also Daggett et al., 1996
). On the other hand,
100 µM RG50864 did not significantly inhibit the laminin-
induced clustering (Fig. 6, striped columns). These findings
indicate that the clustering of DG by agrin and by laminin
involves two different sets of intermolecular linkages.
Agrin and laminin also differed in their action on spontaneously formed megaclusters. In agreement with previous studies on this culture system, AChR staining revealed
that most of the muscle cells in control cultures typically
have one or two megaclusters, usually on their ventral surface (Fig. 11 A). We also confirmed that agrin (Daggett et al.,
1996), like innervation (Anderson and Cohen, 1977
;
Moody-Corbett and Cohen, 1982
), inhibits the formation
and/or survival of these megaclusters (Fig. 11 B). By contrast, the incidence of megaclusters was not reduced by
chronic treatment with laminin (Fig. 11 C). Based on examination of 100 cells/culture, ventral surface megaclusters were seen on 79.5 ± 1.8% (mean ± SEM; n = 17) of
the cells in control cultures compared to only 15.0 ± 7.3%
(n = 7) for cultures treated with agrin for 1 d. The corresponding values for cultures treated with laminin (2.4-6
nM) for 1 d (78.3 ± 4.3%; n = 7) or for 2-3 d (81.3 ± 3.5%; n = 3) were similar to the control value. Thus, unlike agrin-induced clustering, laminin-induced clustering
does not interfere with the occurrence of spontaneously
formed megaclusters.
This study has indicated that treatment with laminin leads
to a clustering of DG on the surface of embryonic muscle
cells. DG consists of the extracellular peripheral membrane protein DG tightly bound to its transmembrane
partner
DG, both of which are derived from the same
precursor protein (Ibraghimov-Beskrovnaya et al., 1992
,
1993; Bowe et al., 1994
; Yoshida et al., 1994
). Laminin,
which in known to bind to
DG (Ibraghimov-Beskrovnaya et al., 1992
; Ervasti and Campbell, 1993
; Gee et al.,1993), is present at these clusters, as is dystrophin, which is known
to bind to
DG (Suzuki et al., 1994
; Jung et al., 1995
). In
contrast to agrin-induced clustering, laminin-induced clustering did not involve AChRs, utrophin, or phosphotyrosine and was not inhibited by the tyrphostin RG50864, a
tyrosine kinase inhibitor. Clustering was apparent within
150 min of treatment with 6 nM laminin, was inhibited by
low temperature, and occurred at concentrations as low as
30 pM, the lowest tested. The laminin-induced increase in
the formation of macroclusters was accompanied by a decrease in the density of microclusters. This inverse relationship implies that the macroclusters were derived from
the microclusters.
On the other hand, it is highly unlikely that the macroclusters were derived from megaclusters. The laminin- induced macroclusters were distributed over the entire dorsal surface of the muscle cells, whereas megaclusters occupied a very limited region of the cell and were situated most often on the ventral surface. Moreover, in contrast to the case for agrin treatment and for innervation, laminin treatment did not stimulate the disappearance of megaclusters. An alternative possibility, that laminin-induced macroclusters resulted simply from an increased incorporation of DG at the cell surface, also seems unlikely in view of the fact that the formation of macroclusters was accompanied by a marked decline in the density of microclusters.
The simplest explanation of these findings is that DG
microclusters on the surface of embryonic muscle cells are
mobile or are unstable and give rise to single, mobile DG
complexes (Fig. 12). In fact, some of the smallest microclusters may actually have been single molecules. In the
absence of laminin, collisions between the mobile DG fail
to result in the formation of stable, long-lived aggregates.
However, as indicated in the model of Fig. 12, when laminin is bound to the mobile DG, stable and hence larger aggregates do form, necessarily resulting in a decline in the
density of the microclusters. Also consistent with this interpretation is the paucity of endogenous laminin (Fig. 3 C;
Vogel et al., 1983) and extracellular matrix (Kullberg et al.,
1977
; Weldon and Cohen, 1979
; Hall and Sanes, 1993
) on
the surface of the embryonic muscle cells. The extensive
clustering at the cell periphery, where the dorsal and ventral surfaces meet, can also be readily explained by this diffusion trap mechanism. Fluorescent staining for laminin, after briefly exposing culture chambers to laminin at
higher concentrations than used in the present study, reveals that laminin binds tightly to the culture substrate
(Cohen, M.W., unpublished observations). Accordingly,
when mobile laminin-DG complexes on the dorsal surface happen to reach the cell periphery and contact the culture
substrate, they become at least partially immobilized. The
presence of this additional trapping factor at the cell periphery necessarily leads to an extensive clustering at this site.
Our observations with the laminin fragment E3 provide
additional insight into the mechanism of the laminin-
induced clustering. E3 consists of the last two G repeats at
the end of laminin's long arm (Yurchenco and Cheng,
1993) and competes with laminin for binding to
DG (Gee
et al., 1993
). Since E3 inhibited the laminin-induced clustering, it follows that laminin's
DG binding domain is required for clustering DG. Furthermore, E3 itself did not
exhibit any clustering activity, thereby indicating that other portions of the laminin molecule are also required.
The possible identity of these other portions is suggested by studies on the aggregation of laminin molecules
in solution. It has been found that this aggregation involves
self binding domains at the ends of the short arms of the
laminin molecule (Yurchenco and Cheng, 1993; Colognato-Pyke et al., 1995
). These self binding domains are obvious candidates for participation in the clustering of mobile laminin-DG complexes. At first glance, one apparent
inconsistency is that aggregation of laminin in solution occurs at a critical concentration of ~60 nM (Yurchenco
and Schittny, 1990
), whereas the critical concentration for
laminin-induced clustering on embryonic muscle cells was
some 1,000 fold lower. However, at least part of this difference in critical concentrations can be explained by the
alignment of laminin's self binding domains when it is
bound to
DG (Fig. 12) and by the restriction of movement to two dimensions rather than three. Under such conditions a much greater proportion of the laminin-laminin
collisions would involve the self binding domains than would
be the case for unbound laminin molecules in solution. In
fact, laminin aggregates into macroclusters when it binds
to synthetic lipid bilayers, and the critical concentration for this aggregation is about an order of magnitude lower
than self aggregation in solution (Kalb and Engel, 1991
).
The probability of laminin-laminin binding might be increased even further by the interaction of mobile laminin-
DG complexes with other molecules. One such molecule is
dystrophin, which was found to be present at the laminininduced clusters presumably bound to DG. Since dystrophin can bind to cytoskeletal actin (Hemmings et al., 1992
)
it would be expected to facilitate the aggregation of colliding laminin-DG complexes. In the absence of laminin,
however, this interaction by itself is apparently too weak
to promote the formation of large, stable aggregates between colliding DG molecules. This would account for the
relatively low incidence of macroclusters and the high density of microclusters in control cultures.
That cluster formation on the surface of embryonic muscle cells involves an aggregation of preexisting mobile
membrane proteins has been established for nerve- and
agrin-induced clustering of AChRs (Anderson and Cohen,
1977; Godfrey et al., 1984
; Ziskind-Conhaim et al., 1984
;
Kidokoro et al., 1986
). The present study supports the proposal (Cohen et al., 1995a
) that the DG, which accumulates together with AChRs at these clusters, is also derived
from a preexisting pool of mobile DG. While recruitment
of mobile DG may be common to both agrin- and laminininduced clustering, the molecular interactions responsible
for trapping DG are clearly different. Agrin's action depends on activation of the transmembrane receptor tyrosine kinase MuSK (DeChiara et al., 1996
; Glass et al., 1996
),
although the identity of the tyrosine phosphorylated intermediates which promote the trapping of DG and AChRs
at the same site is unclear. By contrast, laminin-induced
clustering does not appear to involve tyrosine kinase activation or the participation of tyrosine phosphorylated proteins. Instead, as discussed above, laminin-laminin binding
may be the key molecular interaction involved in the trapping of mobile laminin-DG complexes.
Based on this diffusion trap scheme, other surface molecules which bind laminin, such as integrins and perlecan
(Timpl and Brown, 1994), might also be expected to participate in laminin-induced clustering. Where such molecules
are already anchored they could act as foci for trapping
mobile laminin-DG complexes. Where these other lamininbinding molecules are themselves mobile, laminin could
cluster them together with DG. It might be expected as well that different isoforms of laminin (Timpl and Brown, 1994
)
will have different clustering activities from the laminin-1
isoform studied here.
Another difference between the action of agrin and laminin is that utrophin, like AChRs and PY, accumulates only
at agrin-induced clusters. Both utrophin and dystrophin
have homologous domains for binding to DG (Ozawa et al.,
1995
), yet only dystrophin accumulated at the laminin-
induced clusters. If there are preexisting populations of
DG-dystrophin and DG-utrophin complexes on the muscle cell surface, then laminin may selectively recruit the
former, whereas agrin may selectively recruit the latter.
Alternatively, laminin and agrin may differentially promote the formation of these two different complexes.
Other molecules which are preferentially associated with
agrin-induced clusters (e.g., tyrosine phosphorylated proteins) may also contribute to the accumulation of utrophin by binding it directly or by facilitating its binding to
DG. In this regard it is interesting to note that utrophin is concentrated together with AChRs, DG, and phosphotyrosine at spontaneously formed megaclusters (Cohen,
M.W., unpublished observations) even though agrin is not
detected at these sites (Cohen and Godfrey, 1992
). This
lends support to the suggestion that molecules in addition
to agrin play a role in promoting utrophin accumulation at
AChR-containing clusters.
Whereas treatment with agrin resulted in a marked reduction in the incidence of spontaneously formed megaclusters, treatment with laminin did not. Previous studies
have indicated that the formation and survival of spontaneously formed, AChR-containing megaclusters is inhibited
when there is extensive induction of new AChR-containing clusters elsewhere on the muscle cell surface. The latter relationship has been noted for AChR clustering
induced by innervation (Anderson and Cohen, 1977;
Moody-Corbett and Cohen, 1982
), by application of appropriately coated beads (Peng et al., 1981
; Peng, 1986
), by
pathways of substrate-bound, endogenous neural agrin (Cohen et al., 1995b
), and by diffusely applied agrin (Daggett et
al., 1996
; present study). The mechanism of this competition between newly induced and spontaneously formed
AChR-containing clusters remains to be elucidated but
presumably involves some of the intracellular signalling
events associated with AChR clustering. The present results suggest that laminin's AChR-clustering activity is insufficient to initiate this competition in cultures of Xenopus muscle cells. Only a small and variable increase in
AChR-containing clusters was observed (Table I), consistent with the small (twofold) laminin-induced increase
previously observed on rat myotubes in culture (Vogel et
al., 1983
). The results also indicate that although spontaneously formed megaclusters contain DG, extensive clustering of DG elsewhere on the cell surface does not interfere
with their formation and/or survival.
Laminin-induced clustering occurred at a laminin concentration of 30 pM and probably occurs at even lower
concentrations. Such concentrations may be generated in
vivo when developing cells secrete laminin into a confined
extracellular space. The resulting changes in the distribution of surface molecules to which laminin binds may be
important in the assembly of extracellular matrix and
could have developmental consequences. In this regard it
is interesting to note that the molecular basis of muscular
dystrophy in the dy2J mouse has been ascribed to partial
deletion of one of the self binding domains of laminin-2
(Xu et al., 1994).
Received for publication 15 August 1996 and in revised form 15 November 1996.
D. McDonald, M. Ignatova, and T. Inoue provided excellent technical assistance. We thank K.P. Campbell for the generous gift of anti-This work was supported by grants to M.W. Cohen from the Medical Research Council of Canada, to P.D. Yurchenco from National Institutes of Health (DK36425), to G.E. Morris from the Muscular Dystrophy Group of Great Britain and Northern Ireland, and to S. Carbonetto from the Muscular Dystrophy Association (USA) and the Canadian Centres of Excellence.
AChR, acetylcholine receptor; DAP, dystrophin associated protein; DG, dystroglycan; LBS, laminin binding sites; PY, phosphotyrosine; ROI, region of interest.