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Originally published In Press as doi:10.1074/jbc.M600912200 on March 10, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13365-13373, May 12, 2006
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An Extracellular Pathway for Dystroglycan Function in Acetylcholine Receptor Aggregation and Laminin Deposition in Skeletal Myotubes*

Mathieu R. Tremblay{ddagger} and Salvatore Carbonetto{ddagger}§1

From the {ddagger}Department of Biology, §Center for Research in Neuroscience, McGill University, Montréal General Hospital Research Institute, Montréal, Québec H3G 1A4, Canada

Received for publication, January 30, 2006 , and in revised form, March 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The dystroglycan (DG) complex is involved in agrin-induced acetylcholine receptor clustering downstream of muscle-specific kinase where it regulates the stability of acetylcholine receptor aggregates as well as assembly of the synaptic basement membrane. We have previously proposed that this entails coordinate extracellular and intracellular interactions of its two subunits, {alpha}- and beta-DG. To assess the contribution of the extracellular and intracellular portions of DG, we have used adenoviruses to express full-length and deletion mutants of beta-DG in myotubes derived from wild-type embryonic stem cells or from cells null for DG. We show that {alpha}-DG is properly glycosylated and targeted to the myotube surface in the absence of beta-DG. Extracellular interactions of DG modulate the size and the microcluster density of agrin-induced acetylcholine receptor aggregates and are responsible for targeting laminin to these clusters. Thus, the association of {alpha}- and beta-DG in skeletal muscle may coordinate independent roles in signaling. We discuss how DG may regulate synapses through extracellular signaling functions of its {alpha} subunit.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dystroglycan (DG)2 is a member of the dystrophin-associated glycoprotein complex (DGC). It is encoded by a single gene (dag1) and is expressed initially as a propeptide that gets cleaved to generate two distinct subunits as follows: {alpha}-dystroglycan ({alpha}-DG), a peripheral mucin-like protein, and beta-dystroglycan (beta-DG), a transmembrane protein (1, 2). In mature skeletal muscle, both subunits associate noncovalently and localize to the plasma membrane where they form the functional core of the DGC. Dystroglycan is crucial for muscle fiber integrity and survival and is widely thought to act as a structural element of the cell surface by linking the extracellular matrix (ECM) to the cytoskeleton to protect the cell against the stress of contractions (3, 4). Other studies suggest that DG mediates intracellular signaling in noncontractile cells (5, 6) and can regulate cell death (7, 8), replication (9), and polarity (10, 11).

In skeletal muscle, the absence of DG leads to muscular dystrophy (12, 13). At neuromuscular junctions (NMJs), DG associates directly with rapsyn (14) and is colocalized with acetylcholine receptors (AChRs) (15) by their movement to nascent synapses (16). DG-deficient muscle fibers in vivo and in vitro show diffuse and unstable aggregates of AChRs (12, 17, 18), indicating a role for DG in the condensation and stabilization of AChR microclusters. In addition, laminin, perlecan, and acetylcholinesterase (AChE) are greatly reduced at the NMJs of DGdeficient muscle (12, 18).

The NMJ represents a highly specialized compartment within the multinucleated myotube that extends from the nerve terminal through the intervening synaptic basement membrane and plasma membrane to the underlying cytoskeleton and subsynaptic nucleus (19). Formation of this synaptic compartment is associated with expression, highly restricted localization, and stabilization of molecules. The mechanisms involved are fundamental to our understanding of structural and functional mosaicism within the cells. For example, one of the hallmarks of mature NMJs is the presence of a high density of AChRs (104 receptors/µm2) at the tops of folds in the postsynaptic membrane (20). These receptors are trapped in a region of the plasma membrane directly apposed to the nerve terminal, degrade very slowly (21), and are unable to diffuse within the plasma membrane. Initially, AChR aggregation is a muscle-intrinsic process that does not require innervation (22, 23), but reorganization of the AChRs within the membrane is regulated by secretion from the motor nerve of agrin, a heparan-sulfate proteoglycan (24). Agrin acts via MuSK, a muscle-specific receptor tyrosine kinase, together with a myotube-associated specific coreceptor within the postsynaptic membrane (25). Phosphorylation of MuSK recruits rapsyn, which is closely associated with the major cytoplasmic loop of the AChRs (26-31) and can self-associate to form microclusters of AChRs (27, 32). Activation of MuSK leads to the phosphorylation of the beta subunit of the AChR (33), an important requirement for enhanced linkage of AChRs to the cytoskeleton (34). As a specialized domain of skeletal myofibers, the NMJ offers insights into DG functions in fundamental cellular processes. For example, DG functions in basement membrane assembly by anchoring AChE via interactions with perlecan (18, 35, 36).

In previous studies, we have shown that the glycosylation of {alpha}-DG is regulated by the nerve and that it functions in NMJ formation (37). Also, we proposed that {alpha}- and beta-DG function coordinately to assemble an extracellular and intracellular matrix of proteins (38), which is consistent with a recent report suggesting novel extracellular interactions for DG (39). In this study, we have taken advantage of the well defined role of DG at NMJs to determine whether the extracellular domains of DG function independently in AChR aggregation. We describe here that the functions of DG in AChR aggregation and basement membrane assembly in myotubes can be rescued by viral expression of full-length DG in DG-/- myotube cultures. Interestingly, DG constructs consisting of {alpha}-DG alone or of {alpha}-DG and the extracellular regions of beta-DG can regulate aspects of AChR aggregation, namely the size of AChR aggregates, the distribution of microclusters within them, and the assembly of laminin at these aggregates. The implications of these observations for synapse formation and signaling via DG are discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Dystroglycan cDNA Constructs—Dystroglycan cDNA constructs (Fig. 1A) were generated by PCR amplification using specific oligonucleotides for mouse dag1. PCR products were subcloned inframe into the peGFP-N1 expression vector (Clontech) to generate eGFP fusion transcripts. The CD4 ectodomain/transmembrane sequence expression plasmid was obtained from Dr. R. Dunn (McGill University). Constructs {alpha}-DG and {alpha}/{Delta}ectobetaDG were obtained from Dr. S. Winder (University of Sheffield, UK). All constructs were sequenced for validation (Genome Center, McGill University).

Generation of Recombinant Adenoviruses—Replication-defective adenoviruses were generated using the AdEasy system (Qbiogene) as described by the manufacturer. In brief, eGFP, CD4-eGFP, DG, {alpha}-DG, {Delta}cytoDG, and {alpha}/{Delta}ectobetaDG cDNA fusion constructs were subcloned into the multiple cloning site of pShuttle-CMV. All vectors were linearized using PmeI, and cotransfected with pAdEasy-1 into the BJ5183 electrocompetent E. coli strain. Recombinants were screened by colony size and by restriction digests as described (40). Recombinants were linearized using PacI, purified, and transfected into HEK293 cells using Lipofectamine (Invitrogen). Adenovirus production was observed by plaque formation, and particles were isolated from cellular lysates and amplified through several rounds of infections in HEK293 cells. Titers were determined by TCID50 infection test as described by the manufacturer (Qbiogene).

Cell Culture and Adenoviral Infections—DG+/+ and DG-/- embryonic stem (ES) cells were generated previously (12). The cells were maintained undifferentiated on a layer of mitomycin (Sigma)-treated neomycin-resistant STO feeder cells (ATCC) in culture medium consisting of 80% Dulbecco's modified Eagle's medium-high glucose (Invitrogen), 20% fetal bovine serum (Wisent), 1% penicillin/streptomycin (Invitrogen), 7 x 10-4 % beta-mercaptoethanol (Sigma), and 1000 units per ml of leukemia inhibitory factor (Chemicon). For differentiation, ES and feeder cells were harvested using 0.25% trypsin-EDTA (Invitrogen), resuspended in fresh growth medium, and plated twice on 0.1% gelatin-coated tissue culture dishes for 1 h (preplating steps) to remove feeder cells. ES cells were resuspended in differentiation medium made of 85% Dulbecco's modified Eagle's medium-high glucose (Invitrogen), 15% horse serum (Wisent), 1% penicillin/streptomycin (Invitrogen), and 1% dimethyl sulfoxate (Me2SO; Sigma). Cells were counted and seeded on the inside lid of bacterial Petri dishes in 20-µl drops containing 800 cells. They were grown for 4 days at 37 ° in these hanging drops after which they were transferred to 0.1% gelatin-coated dishes in differentiation medium containing 1% insulin/transferrin/selenium (Invitrogen) for 20-25 days. ES cell cultures were infected at day 15-20 with 108 adenoviral plaque-forming units for 16 h in differentiation medium.

Quantification of AChR Aggregation—Soluble recombinant rat agrin was a generous gift from Dr. M. Ferns (University of California, Davis). To assay AChR clustering in ES cells, differentiated cultures (20-25 days old) were treated with 0.5 nM agrin for 16 h. Cells were washed twice in Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen) and incubated with 2 µg/ml rhodamine-conjugated {alpha}-bungarotoxin (Molecular Probes) for 20 min at 25 °. Cells were washed twice with DPBS and fixed with 2% paraformaldehyde/PBS at 37 ° for 20 min. Cultures were mounted on glass coverslips using ImmunoFloure mounting medium (ICN) and observed with a Zeiss Axioskop fluorescence microscope. Digital images observed with a x63 Zeiss objective were captured with a QImaging Retiga 1300 10-bit digital camera under normalized exposures and quantifications of cluster number, size, and density were generated using Northern Eclipse software (Empix). For confocal images, cultures were observed using a Leica DM LFSA microscope equipped with an Ultraview confocal scanner (PerkinElmer Life Sciences), and images were captured and processed using Metamorph software (Universal Imaging). For cells infected with adenovirus, AChR aggregate size was determined by circling each aggregate with a trace from the free hand tool and by measuring total area as calibrated for the objective used (Fig. 2B). AChR aggregate density was determined as a percentage of total aggregate area (18, 41), where the area of each aggregate that was labeled with {alpha}-bungarotoxin was highlighted using the threshold function (values of 18-255 pixels), and the highlighted area was determined within the traced area (Fig. 2B). AChR aggregate numbers were determined by counting the number of aggregates present in each microscopic field observed. At least 30 nonoverlapping fields were visualized for each set of cultures, and data were collected from at least three separate experiments. For statistical analyses, we normalized the data obtained as described above for AChR aggregate size and density by a logarithmic transformation, and we then performed multiple comparisons using unbalanced one-way analysis of variance followed by pairwise comparisons (Tukey-Kramer and Fisher's tests) on the normalized data. All probability (p) values stated in the text are for analysis on normalized values. All analyses and percentile plots were generated using StatView 4.5 package (Abacus) or SAS (SPSS). All images and figures were prepared using Adobe Photoshop and Illustrator.

Immunocytochemistry—Cultures were stained with {alpha}-bungarotoxin as described above and then fixed in 2% paraformaldehyde/DPBS at 37 ° for 20 min. If applicable, cells were permeabilized in 0.5% Triton X-100 in DPBS for 10 min at 25 °. Cells were blocked with 10% donkey or horse serum at 25 ° for 1 h. This was followed by incubation with either monoclonal antibody IIH6 to {alpha}-DG (1:50) (Upstate Biotechnologies, Inc.), rabbit anti-sera to laminin (homemade, 1:100), anti-GFP antibody (Living Colors; 1:50) (Clontech), or rabbit anti-sera to beta-DG (homemade, 1:100) at 25 ° for 1 h. Cells were washed extensively with DPBS and incubated with rhodamine-conjugated goat anti-mouse IgM (1:100), aminomethylcoumarin acetate-conjugated donkey anti-rabbit (1:100), or rhodamine-conjugated donkey anti-rabbit IgG (1:100) secondary antibodies (Jackson ImmunoResearch), at 25 ° for 1 h. Following a second series of washing, cells were mounted on glass coverslips and analyzed as described above. For laminin-AChR overlap observations, we scored positive only the aggregates that discretely overlapped with laminin immunoreactivity. We normalized to the total number of clusters observed in each case. A minimum of 20 nonoverlapping fields was scored in three experiments for each set of cultures, and differences were analyzed by analysis of variance followed by Fisher's test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A simple model for DG function predicts that extracellular interactions mediated by {alpha}-DG would in turn signal intracellularly via beta-DG. This model is complicated by the fact that most ligands for {alpha}-DG bind to an extended carbohydrate side chain (for review see Ref. 42), and transmembrane signaling would require the unprecedented translation of this interaction into conformational changes in both {alpha}- and beta-DG. Considering that interactions of the {alpha} subunit are extracellular and of the beta subunit are intracellular, we suggest to determine whether {alpha}- and beta-DG might have independent functions in organizing cell surface proteins by generating a series of DG cDNA constructs bearing deletions of beta-DG (Fig. 1). We fused these cDNA constructs at their C terminus to the enhanced green fluorescent protein (eGFP; see "Experimental Procedures"). In this study, we focused on constructs that reveal distinct functions of {alpha}-DG versus full-length DG (Fig. 1). Construct DG encodes full-length {alpha}- and beta-DG subunits; construct {Delta}cytoDG lacks all cytoplasmic domains of beta-DG; construct {alpha}-DG encodes the {alpha} subunit of DG; construct {Delta}ectobetaDG lacks the extracellular domains of beta-DG leaving its transmembrane region following the C-terminal end of {alpha}-DG. All constructs were transiently expressed by infecting muscle cells derived from DG-/- (dag1) ES cells (12, 18) using serotype Ad5 replication-defective adenoviruses under the CMV promoter (see "Experimental Procedures"). DG-/- cells form diffuse aggregates of AChRs in response to agrin (Fig. 2A), and the area occupied by the AChR microclusters as well as their density were quantified by computer-assisted image analysis (Fig. 2B) (18).


Figure 1
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  		FIGURE 1.
Mouse dystroglycan cDNA constructs expressed by adenoviruses. Mouse dystroglycan contains several functional domains as follows: a signal sequence (SS; positions 1-29), a mucin-like region (positions 315-484), a cleavage site (CS, position 653), a type 1 transmembrane region (TM, positions 751-774), a juxtamembrane region (JM), and a C-terminal consensus sequence (PPxY) for interactions with WW domain-containing proteins (PPxY). In addition to putative O-linked carbohydrate sites within the mucin-like region, mouse dystroglycan contains four potential N-glycosylation sites (positions 139, 639, 647, and 659; marked by *). All dystroglycan cDNA constructs were fused to eGFP and were expressed under the CMV promoter. DG, full-length dystroglycan; {Delta}cytoDG, deletion of cytoplasmic sequences of beta-dystroglycan; {alpha}-DG, {alpha}-dystroglycan alone; {alpha}/{Delta}ecto-betaDG, deletion of extracellular sequences of beta-dystroglycan.

 
Expression of DG Constructs in Myotubes Derived from DG-/- ES Cells—Infection of agrin-treated myotubes with DG-expressing adenoviruses resulted in a diffuse intracellular accumulation of the eGFP-tagged constructs (Fig. 3, A-L), a consequence of the constitutive nature of the promoter. To assess the expression of these constructs at the cell surface in infected myotubes, we immunolabeled cultures using three antibodies as follows: monoclonal antibody (mAb) IIH6 against glycosylated {alpha}-DG on nonpermeabilized cultures (43) to visualize cell surface expression; a rabbit antiserum to the last 15 amino acids of beta-DG (38) on permeabilized cultures; and an anti-serum to eGFP. DG+/+ cells infected with a control eGFP-expressing adenovirus (Fig. 3, A and B) showed regions of aggregated surface labeling of {alpha}-DG (Fig. 3A') and of beta-DG (Fig. 3B') in a distribution that is identical to uninfected DG+/+ cells (data not shown). As expected, DG-/- cells infected with eGFP (Fig. 3, C and D) expressed neither {alpha}-DG (Fig. 3C') nor beta-DG (Fig. 3D'). Expression of full-length DG in DG-/- cells (Fig. 3, E and F) showed aggregates of {alpha}-DG (Fig. 3E') and beta-DG (Fig. 3F') similar to that in DG+/+ cells (Fig. 3, A' and B'). Our immunofluorescence analysis indicated approximately equivalent levels of expression of DG constructs in DG-/- cells when compared with endogenous levels in DG+/+ cells. Expression of {Delta}cytoDG (Fig. 3, G and H) resulted in surface expression of this construct which encompasses both {alpha}-DG (Fig. 3G') and the extracellular and transmembrane regions of beta-DG as detected by anti-eGFP (Fig. 3H'). In this instance, labeling with mAb IIH6, which recognizes a functional carbohydrate epitope on {alpha}-DG, indicated that proper glycosylation of {alpha}-DG does not require beta-DG cytoplasmic domains. Expression of {alpha}-DG lacking any anchor to the cell surface via beta-DG (Fig. 3, I and J) also resulted in its surface deposition on myotubes as detected by both IIH6 and anti-GFP antibodies in nonpermeabilized cells (Fig. 3, I' and J'). The distribution of this {alpha}-DG construct on DG-/- cells was similar to the endogenous {alpha}-DG expression observed on DG+/+ cells (Fig. 3A'). {alpha}-DG was not readily detected on the surface of infected non-muscle cells (data not shown), suggesting that {alpha}-DG, when expressed alone, preferably bound to muscle cells. The heterogeneity of ES-derived cell cultures and their low abundance in myotubes prevented us from assessing glycosylation or expression of {alpha}-DG by Western blot analysis. Nonetheless, in another study, we determined that expression of {alpha}-DG alone in adenovirus-infected COS cells resulted in its secretion into the culture medium (data not shown), and it could be identified by Western blotting using mAb IIH6 (data not shown). Finally, expression of construct {alpha}/{Delta}ectobetaDG (Fig. 3, K and L) showed surface labeling of {alpha}-DG (Fig. 3K') and beta-DG (Fig. 3L') as observed for endogenous or ectopic full-length DG (Fig. 3, E' and F'). Taken together, these observations show that DG cDNA constructs could be expressed in myotubes derived from DG-/- ES cells using adenoviruses and that both {alpha}- and beta-DG were normally expressed at the cell surface. Proper surface targeting and glycosylation of {alpha}-DG could occur in the absence of beta-DG expression. Moreover, the association of a form of {alpha}-DG lacking any anchorage to beta-DG still was able to interact with cell surface proteins either in the ECM or in the plasma membrane possibly affecting interactions and functions of the DG complex.


Figure 2
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  		FIGURE 2.
Agrin-induced AChR aggregate morphology in myotubes derived from ES cells. A, myotubes derived from DG+/+ and DG-/- ES cells were treated with 0.5 nM soluble agrin and labeled with rhodamine-conjugated {alpha}-bungarotoxin. DG+/+ myotubes form small condensed aggregates of AChRs (left panel), and DG-/- myotubes display disrupted aggregates reflecting a collection of microclusters (middle panel). Expression of a membrane-bound eGFP (CD4eGFP) in DG-/- myotubes does not alter the distribution of AChR microclusters (right panel). Pictures show a single aggregate for each myotube. B, methods for measuring size and density of AChR aggregates on myotubes. Using Northern Eclipse software (version 6.0, Empix), captured images of bungarotoxin-labeled AChR aggregates were inverted and calibrated for the x63 Zeiss objective used; using the freehand tool, aggregates were encircled, and the area was measured from the "measure" command to evaluate the total aggregate area (upper right panel). The same aggregate was processed using the threshold tool to highlight the AChR microclusters labeled within each aggregate (lower left panel). The area from the highlighted AChRs was measured. To express the relative distribution of AChRs within the aggregate (density), we used the ratio between the measured value for area of AChR microclusters within aggregates to the measured value for total aggregate size (lower right panel). Bar, 15 µm.

 


Figure 3
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  		FIGURE 3.
Expression of DG in agrin-treated muscle fibers differentiated from ES cells. DG+/+ (A, A', B, B') or DG-/- (C-L, C'-L') myotubes were infected with different adenoviral constructs (Ad; A-D, eGFP control; E and F, DG; G and H, {Delta}cytoDG; I and J, {alpha}-DG; K and L, {alpha}/{Delta}ectobetaDG), treated with agrin, and immunolabeled using mAb IIH6 (A', C', E', G', I', and K', {alpha}DG), antisera to beta-DG (B', D', F', and L', betaDG), or anti-GFP antibody (H' and J', GFP). Arrows point to typical surface labeling. Bar, 50 µm.

 
Expression of Dystroglycan in DG-/- Myotubes Restores the Size of AChR Aggregates—Myotubes derived from both DG+/+ and DG-/- ES cells express AChRs and respond to soluble agrin by forming aggregates of receptors on their surfaces (17, 18), indicating that DG is downstream of the agrin/MuSK signaling pathway (44). However, in the absence of DG, these agrin-induced aggregates are an abnormally large, diffuse, and unstable collection of microclusters of AChRs (Fig. 2A) (18). Furthermore, these DG-deficient aggregates lack laminin, merosin, perlecan, and AChE (18). To ascertain whether {alpha}- and beta-DG act only as a complex in response to MuSK activation, we infected DG-/- myotubes with adenoviruses expressing either eGFP or DG constructs (Fig. 1), and 5-7-day post-infection, treated the cultures with a saturating concentration (0.5 nM) of soluble agrin to induce AChR aggregation at the surface of myotubes (45), and labeled AChRs using rhodamine-conjugated {alpha}-bungarotoxin. As controls for the assays, we used soluble cytoplasmic eGFP or submembrane eGFP (CD4-eGFP). This latter construct allowed us to control for any effect due to membrane expression of eGFP on AChR aggregation. Expression of either control constructs in DG+/+ or DG-/- cells did not affect the aggregation of AChRs after agrin treatment (Figs. 2A and 4).

After agrin treatment, DG+/+ myotubes infected with eGFP had several small, tight aggregates of AChRs (Fig. 4) with about 85% of aggregates below 100 µm2 (mean size, 45.8 ± 5.0 µm2; see Fig. 5). DG-/- myotubes expressing eGFP responded to agrin, but the resulting aggregates were more diffuse, punctate, and significantly larger (Figs. 2A and 4, inset) with 50% or more of the aggregates above 100 µm2 (mean size, 121.5 ± 9.9 µm2; p < 0.0001; see Fig. 5). Essentially identical results were found in DG+/+ and DG-/- cells uninfected with adenoviruses (Fig. 2A and data not shown) (18) or in myotubes expressing CD4-eGFP (Fig. 2A) (mean size, 101.0 ± 8.1 µm2; p < 0.05). When DG-/- cells were infected with an adenovirus expressing full-length DG, AChR aggregates were smaller and essentially identical to those in DG+/+ cells (Fig. 4). About 80% of the aggregates were below 100 µm2 (mean, 49.5 ± 4.9 µm2; see Fig. 5), were not significantly different in size from DG+/+ cells (p = 0.7787), and were clearly smaller than those on DG-/- cells expressing either eGFP or CD4-eGFP controls (p < 0.0001). Thus, expression of full-length DG in DG-/- myotubes was capable of rescuing AChR aggregate size to that seen in DG+/+ myotubes.


Figure 4
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  		FIGURE 4.
Agrin-induced AChR aggregates in adenovirus-infected myotubes derived from ES cells. DG+/+ and DG-/- myotubes differentiated from ES cells were infected with corresponding adenoviruses, treated with recombinant agrin for 16 h, and labeled for AChRs using rhodamine-conjugated {alpha}-bungarotoxin. Arrows and insets indicate representative AChR aggregates in each case. Bar, 50 µm.

 


Figure 5
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  		FIGURE 5.
Dystroglycan affects agrin-induced AChR aggregate size. Relative frequency distribution for the different populations of AChR aggregates observed in adenovirus-infected myotube cultures. Measures are in microns (µm2) and frequencies in percentages (%).

 
To assess the relative contribution of extracellular and intracellular interactions of DG to AChR aggregation, DG-/- cells were infected with adenoviruses expressing either {Delta}cytoDG, {alpha}-DG, or {alpha}/{Delta}ectobetaDG (Figs. 3 and 4) and treated with soluble agrin. Expression of {Delta}cytoDG, which includes full-length {alpha}-DG and the extracellular domains of beta-DG to which it binds, in DG-/- cells (Fig. 4) resulted in a highly significant but partial reduction in aggregate size (mean, 96.9 ± 6.3 µm2) when compared with DG-/-:eGFP (p < 0.0005; see Fig. 5). Expression of {alpha}-DG also resulted in reduction of aggregate size (mean, 77.6 ± 4.2 µm2) when compared with DG-/-:eGFP (p < 0.0001; see Fig. 5). Interestingly, expression of {alpha}/{Delta}ectobetaDG resulted in full recovery of AChR aggregate size (mean size, 47.8 ± 2.9 µm2) when compared with DG-/-:eGFP (p < 0.0001) and similar to the expression of endogenous or ectopic full-length DG. We did not observe any increase in numbers of AChR aggregates per myotube after expression of the different DG constructs in DG-/- cells (data not shown). These observations suggest that interactions mediated via extracellular regions of DG can function to regulate AChR aggregate size. Moreover, expression of {alpha}-DG and of its beta-DG-binding sites can function as well as the noncovalently associated complex of {alpha}- and beta-DG.

Dystroglycan Regulates the Distribution of AChR Microclusters within Aggregates—In cultured myotubes, agrin in solution (46, 47) or released by the nerve (48) stimulates the formation of microclusters of AChRs, which subsequently condense into a mature postsynaptic plaque. Myotubes deficient for DG fail to form such plaques and as a result the area of an aggregate occupied by AChRs is greater and the microclusters remain distinct puncta (Fig. 2A). Because full-length or extracellular DG regulate aggregate size (see above), we asked whether extracellular interactions of DG were also capable of controlling AChR packing density of microclusters within agrin-induced AChR aggregates. In this case, we defined packing density as the relative area of an aggregate covered by AChR microclusters labeled with fluorescent {alpha}-bungarotoxin (Fig. 2B) (18, 41). When quantified in myotubes derived from DG+/+ cells treated with agrin, this packing density yielded a frequency histogram with a normal distribution and a mean of ~70%. Uninfected or eGFP-expressing DG+/+ cells showed the same mean (66.7 ± 2.0%; see Fig. 6), but uninfected or eGFP-expressing DG-/- cells had a significantly lower relative area occupied by microclusters (mean, 45.1 ± 2.8%; p < 0.0001). Expressing full-length DG in DG-/- cells clearly increased the relative area occupied by microclusters with a mean of 56.7 ± 1.9% when compared with DG-/-:eGFP (p = 0.022), but it remained somewhat lower than in DG+/+:eGFP cells (p = 0.0474; see Fig. 6). Thus, expression of full-length DG significantly but not completely rescued the normal distribution of microclusters within aggregates on myotubes derived from DG-/- ES cells to produce a density of AChRs approximately that in DG+/+ cells. Interestingly, expression of {Delta}cytoDG in DG-/- cells also significantly increased the area occupied by microclusters (mean, 60.2 ± 1.5%) when compared with DG-/-:eGFP (p = 0.005; see Fig. 6), suggesting that the extracellular domains of DG are responsible for most if not all of the activity of full-length DG in regulating AChR density. We observed a similar significant increase when either {alpha}-DG (mean, 60.8 ± 2.2%; p < 0.0001) or {alpha}/{Delta}ectobetaDG was expressed (mean, 65.7 ± 2.0%; p < 0.0001). When the populations are ranked by percentiles (Fig. 6B), the distribution plot for DG-/-:{Delta}cytoDG cells looked similar to that for DG+/+:eGFP or DG-/-:DG cells but was distinct from control DG-/-:eGFP cells. In contrast, the plots for DG-/-:{alpha}-DG and DG-/-:{alpha}/{Delta}ectobetaDG cells showed a greater variation across the percentiles. Together, these data suggest that DG regulates the distribution of microclusters of AChRs within aggregates induced by agrin, and extracellular interactions via {alpha}-DG function to achieve this regulation.

Localization of Dystroglycan to AChR Aggregates—Dystroglycan interacts with rapsyn (14) and localizes precisely to nascent aggregates of agrin-induced AChRs on skeletal myofibers in vivo (16). However, in C2C12 myotubes treated with agrin, DG accumulates in most but not all AChR aggregates (8, 43). Furthermore, in the same myotubes treated with laminin-1 that can also stimulate AChR aggregation via DG, DG is not colocalized with AChR aggregates (49). Because DG regulates AChR aggregation, we asked whether minimal constructs of DG are localized to these aggregates in DG+/+ and DG-/- myotubes. To assess this, we immunolabeled DG using mAb IIH6 (see Fig. 3) to detect an overlap between surface {alpha}-DG and AChR aggregates (Fig. 7). In DG+/+ cells infected with eGFP, DG was expressed along the myofiber surface and accumulated with several but not all AChR aggregates (Fig. 7, DG+/+:eGFP, arrows). As expected, in DG-/- cells infected with eGFP, there was no DG detected (Fig. 7, DG-/-: eGFP, arrowheads). Only full-length DG expressed in DG-/- cells localized to most AChR aggregates (Fig. 7, DG-/-:DG, arrows) but not all (arrowheads). Expression of {Delta}cytoDG, {alpha}DG, or {alpha}/{Delta}ectobetaDG in DG-/- cells was detected along the myotube surface (Fig. 3) but was seldom found colocalized with AChR aggregates (data not shown). That full-length DG is found more frequently at AChR aggregation may reflect targeting to clusters via interactions with rapsyn. Constructs lacking these domains are not similarly targeted but nevertheless can function in regulating aggregate size and density. Possibly this is mediated by participation of DG in ECM assembly, which at sites of AChR aggregation, even distant ones, can affect AChR aggregation by interactions with MuSK (50) or some proteins essential to the primary AChR scaffold (discussed below).


Figure 6
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  		FIGURE 6.
Dystroglycan modulates agrin-induced AChR aggregate density. A, relative frequency distribution for the different populations of AChR aggregates observed in adenovirus-infected myotube cultures. Densities are expressed as a percentage (%) of aggregate area (see "Experimental Procedures"). B, percentile plots corresponding to the populations in A.

 


Figure 7
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  		FIGURE 7.
Localization of dystroglycan to AChR aggregates in infected myotubes. DG+/+ and DG-/- myotubes were infected with the corresponding adenoviruses, treated with soluble agrin, labeled with rhodamine-conjugated {alpha}-bungarotoxin (left panel, red), and immunolabeled with mAb IIH6 to {alpha}-DG (middle panel, green). Right panel shows merged images. Arrows point to positive overlap (yellow) and arrowheads to negative overlap. Bar, 25 µm.

 
{alpha}-Dystroglycan Is Sufficient to Target Laminin to AChR Aggregates— Laminin is a major component of the muscle basement membrane, and synapse-specific isoforms are targeted to NMJs (51-53). Indeed, laminin induces AChR aggregation (54, 55) via DG (18, 44, 56). Interestingly, laminin-1 fails to accumulate to laminin-induced AChR aggregates in treated C2C12 myotubes (49). Furthermore, in agrin-treated myotubes in culture, about 20% of AChR aggregates do not contain laminin (44, 57). The DG complex functions in basement membrane assembly, and its function is important for some (58, 59) but not all (7) basement membranes. DG is necessary to target laminin, perlecan, and AChE to AChR aggregates and to the synaptic basement membrane (12, 18, 35). These studies suggest that DG is a laminin receptor necessary for basement membrane assembly at select structural and functional domains of cells. Furthermore, the synaptic assembly of laminin at agrin-induced AChR aggregates clearly relies on DG. Hence, we asked whether the expression of DG mutants in DG-/- cells affected the laminin deposition to AChR aggregates. In control DG+/+ cells, 77.7 ± 8.3% of AChR aggregates overlapped with laminin accumulation on the surface of muscle cells (Fig. 8); on the other hand, control DG-/- cells showed only 13.1 ± 4.7% overlap (compared with DG+/+, p = 0.001). Control expression of eGFP in DG-/- cells resulted in 15.8 ± 3.9% overlap (no different from DG-/- control, p = 0.70). Similarly, expression of CD4-eGFP in DG-/- cells did not increase the laminin-AChR overlap (6.5 ± 4.3% overlap, no different from DG-/- control or DG-/-:eGFP, p = 0.97). Expression of full-length DG in DG-/- cells increased the coincidence of laminin with AChR clusters to 69.6 ± 8.6% (compared with DG-/-, DG-/-:eGFP, or DG-/-:CD4eGFP, p < 0.0001), indicating that full-length DG is sufficient for targeting of laminin to AChR aggregates. Expression of {Delta}cytoDG also recovered laminin deposition to AChR aggregates (51.4 ± 7.5%; when compared with DG-/-, DG-/-:eGFP, or DG-/-:CD4eGFP, p < 0.002). Expression of {alpha}DG on the surface of DG-/- cells significantly increased the coincidence of laminin with AChR aggregates (35.9 ± 7.4%; when compared with DG-/-, DG-/-:eGFP, or DG-/-:CD4eGFP, p < 0.05). Finally, expression of {alpha}/{Delta}ectobetaDG in DG-/- cells resulted in a 50.1 ± 6.8% overlap (p = 0.02), similar to the effects observed by {Delta}cytoDG. Taken together, these observations suggest that full-length DG dominates in the regulation of laminin assembly in agrin-induced AChR aggregation. Furthermore, this implies that extracellular interactions via {alpha}-DG can function in the synaptic localization of laminin in myotubes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated previously that {alpha}- and beta-DG are recruited to developing NMJs (16) where they regulate the size and the stability of postsynaptic densities of AChRs and the assembly of a synaptic basement membrane (12, 18). We hypothesized that DG functions via an extracellular as well as an intracellular matrix of proteins (38); consistent with this, a recent report suggests novel extracellular interactions of {alpha}-DG in AChR aggregation (39). Here we show that {alpha}-DG is glycosylated and targeted to the cell surface in absence of beta-DG (Fig. 3). We also show that the DG complex can function extracellularly through {alpha}-DG to regulate the size of AChR aggregates (Figs. 4 and 5) and the density of AChR microclusters within them (Figs. 4 and 6). Moreover, DG constructs lacking either the extracellular or cytoplasmic domains of beta-DG can mediate laminin assembly at agrin-induced AChR aggregates (Fig. 8). We conclude that the {alpha} and beta subunits of the DG complex, which have been widely viewed as two interacting and inter-dependent subunits in skeletal muscle, can function independently (discussed below).


Figure 8
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  		FIGURE 8.
Laminin targeting to AChR aggregates on infected myotubes. A, DG+/+ and DG-/- myotubes were infected with corresponding adenoviruses, labeled with rhodamine-conjugated {alpha}-bungarotoxin (AChRs, right), and immunostained for laminin (Laminin, left). Arrows indicate overlap between laminin and AChRs, and arrowheads point to absence of laminin at AChR aggregates. Asterisks mark accumulation of extra-junctional laminin. B, quantification of discrete laminin overlap with AChR aggregates shown in A. Asterisks denote significant differences with DG-/-:eGFP (p < 0.05). Bar, 25 µm.

 
Processing and Targeting of Dystroglycan in Muscle—Surface expression and glycosylation of DG are crucial for proper function of the DGC in skeletal muscle and in the brain (60, 61). Aberrant glycosylation of {alpha}-DG, due to mutations in glycosylating intermediates, or reduced surface expression of {alpha}-DG, due to lack of dystrophin, leads to the development of muscular dystrophies (60-63). Previous studies from Campbell and co-workers (64) have identified a site for the interaction of Large, an important step in the glycosylation pathway of DG within its {alpha} subunit. Our results extend this by showing that not only is beta-DG unnecessary for this glycosylation but {alpha}-DG can be targeted to the cell surface in the absence of beta-DG (Fig. 3). Indeed, expression of constructs encoding {alpha}-DG and {Delta}cytoDG are detected by mAb IIH6 (Fig. 3), which recognizes an O-linked carbohydrate side chain on {alpha}-DG at the myotube surface that interacts with laminin G domain-containing ligands (65, 66). This indicates that the DG cytoplasmic domain is not necessary for the functional glycosylation of the {alpha} subunit. In fact, this glycosylation may be important in targeting {alpha}-DG to the cell surface, which could be facilitated by the association with basement membrane components, most likely perlecan, agrin, or laminin.

{alpha}-DG contains at its C-terminal end a region highly homologous to a sperm protein, enterokinase, agrin (SEA) module (67) that is found in membrane-tethered mucin proteins (68). In human MUC-1, the SEA module has been shown to undergo autoproteolysis. Point mutations of a conserved serine (Ser-53) residue within the SEA module abrogate this cleavage activity (69). Similarly, in vertebrates, cleavage of DG into {alpha}- and beta-DG depends upon a serine (Ser-655) found in this putative SEA module region (67). However, inhibition of cleavage of DG subunits in point mutants does not prevent the cell surface localization of DG in heterologous cells. These observations raise the question of why vertebrate DG evolved into two subunits. One possibility is to generate distinct functions for the two subunits cleaved from the DG precursor peptide. Noncovalent association between {alpha}- and beta-DG may allow the two subunits to dissociate and possibly interact independently with other partners in the cell or in the ECM. Consistent with this, there are several reports of disjunction in {alpha}- and beta-DG localization in non-muscle tissue (70-72).3

{alpha}-Dystroglycan in AChR Aggregation—Here we have utilized AChR aggregation as a relatively simple and well characterized cellular event that requires DG (12, 17, 18, 44, 73) to study its function in muscle where it is essential for cell integrity in vivo (12) as well as synapse formation. We show that expression of full-length DG or of DG lacking the beta subunit in DG-/- myotubes alters AChR aggregate morphology. Both the size of AChR aggregates and the density of the microclusters within aggregates are the same as those in DG+/+ cells following the expression of DG in DG-/- myotubes (Figs. 4, 5, 6). Expression of DG constructs lacking the beta subunit or its cytoplasmic regions was also sufficient to recover the wild-type morphology of DG-null AChR aggregates (Figs. 4, 5, 6). These observations suggest that {alpha}-DG can act independently from beta-DG in regulating AChR aggregation. The initial interactions of {alpha}-DG must be extracellular, distinguishing it from classical transmembrane signaling. By extension, these data indicate that DG, and more broadly the DGC, may function extracellularly in formation of NMJs. Recent studies have reported that inactive muscle agrin can be potentiated to stimulate AChR aggregation when complexed with laminin into an ECM (57, 74). The association of {alpha}-DG with laminin, agrin, and perlecan may similarly facilitate the assembly of endogenous ligands to stimulate AChR aggregation. More generally, extracellular interactions appear critical for NMJ formation. For example, in Caenorhabditis elegans, LEV-10, a type I transmembrane protein containing Clr/Cls, Uegf, and bone morphogenic protein and low density lipoprotein a domains (75), and CAM-1 (Canal-associated neurons abnormal migration protein 1), a retinoic acid-related orphan receptor tyrosine kinase resembling MuSK (76), are necessary for the aggregation of AChRs at nascent NMJs in a process involving only extracellular interactions. This extracellular signaling may serve to coordinate multiple transmembrane pathways during the assembly of the extremely complex structure of the postsynaptic membrane.

The mechanism of action of DG in AChR aggregation is dependent upon its levels of expression. The absence (12, 18) and excessive levels (77, 78)4 of full-length DG or beta-DG alone in skeletal myotubes lead to aberrant postsynaptic densities of AChRs. In our study, extracellular portions of DG ({alpha}-DG and {Delta}cytoDG) seem to largely dictate AChR aggregate morphology (Figs. 5 and 6). The size of these aggregates on {alpha}-DG- or {Delta}cytoDG-expressing DG-/- cells, although significantly smaller than control DG-/- cells, are larger than in DG-/- cells expressing full-length DG or {alpha}/{Delta}ectobetaDG. The microcluster distribution within these aggregates is the same in these former cell populations. Thus, the total number of microclusters within aggregates on myotubes expressing {alpha}-DG and {Delta}cytoDG is apparently greater than on those expressing full-length DG or {alpha}/{Delta}ectobetaDG. This effect of DG expression could be explained by an overaccumulation of AChR microclusters within aggregates resulting from an altered metabolic turnover (decrease) of AChRs mediated by the extracellular portions of DG. Related to this, Xu and Salpeter (79) have reported that AChR turnover in vivo is increased in mouse muscle lacking dystrophin.

Dystroglycan in Extracellular Signaling—DG is necessary for the formation of some (58) but not all (7) basement membranes. For example, DG is necessary in development for the maintenance of the extra-embryonic Reichert's membrane (80). Deletion of DG from the brain leads to aberrant basement membranes at astroglial end feet on blood vessels and meninges (60). On the other hand, DG-deficient skeletal muscle fibers assemble an ultrastructurally normal basement membrane containing laminin, collagen, fibronectin, and perlecan (12). In our myotube cultures, DG is necessary for the synaptic localization of laminin (Fig. 8) (18). The constructs {alpha}-DG and {Delta}cytoDG clearly target laminin to AChR aggregates (Fig. 8). This suggests that the {alpha}-DG subunit may function as an ECM protein to assemble a basement membrane involved in the regulation of AChR aggregation (Figs. 4, 5, 6) (74) and in the accumulation of laminin at AChR aggregates (Fig. 8).

Extracellular interactions via {alpha}-DG appear to be essential in DG function at NMJs because the disruption of {alpha}-DG interactions with laminin by blocking antibodies (43), with laminin-2 by deletions (57), or by hypoglycosylation of {alpha}-DG, as observed in myd mice (81), leads to aberrant NMJs with disrupted AChR aggregates at the end plate. Also, the idea that the extracellular domains of DG appear sufficient to target and maintain laminin to AChR aggregates (Fig. 8) and to contribute to basement membrane assembly is consistent with observations that laminin binding to sulfatides (6) can seed basement membrane assembly via DG and utrophin and emphasizes the contribution of extracellular interactions of the DGC to this process. In our study, full-length DG and {alpha}/{Delta}ectobetaDG contain the cytoplasmic regions of beta-DG responsible for interaction with Grb2 (growth factor receptor-bound protein 2), rapsyn, caveolin 3, and dystrophin/utrophin (15, 82-84). These regions appear to functionally complement those of the extracellular portion of the DG complex by regulating AChR aggregate morphology (Figs. 4, 5, 6) and basement membrane assembly (Fig. 8).

Our results raise the question of how extracellular domains of DG function in skeletal muscle in AChR aggregation in ways often attributed largely to direct or indirect interactions of AChRs with cytoskeletal proteins. Interactions of matrix proteins have the potential to introduce a level of extracellular protein-protein interactions that eventually signals into the cell interior via several mechanisms. These may include functions of glycosylated {alpha}-DG in signaling cross-talk with integrins; laminin bound to DG could bind via distinct sites to {alpha}6 or {alpha}7 integrins (7, 57) in muscle to signal intracellularly. Other mechanisms could involve perlecan, a ligand for {alpha}-DG and a well known coreceptor for FGF2 (85, 86); neuregulin-1, which regulates AChR synthesis and is a growth factor/ECM protein that is bound to heparan sulfate proteoglycans (87); or AChE, which binds to {alpha}-DG via perlecan (35, 36) and interacts directly with MuSK to alter its distribution (50). Similar extracellular interactions may occur in central nervous system synapses where DG is expressed in neurons of the cerebral cortex and hippocampus (88) and has been implicated in long term potentiation (61) and in synaptic transmission in the retina (89). Conceivably, DG and the DGC may regulate the aggregation of neurotransmitter receptors by virtue of their stabilization in the plasma membrane in a fashion similar to that at NMJs. Further studies to elucidate the mechanisms of DG function in cell survival and synapse formation may contribute to our understanding of the muscle wasting and mental retardation associated with muscular dystrophies.


   FOOTNOTES
 
* This work was supported by a doctoral research award from Canadian Institutes of Health Research (to M. R. T.) and by grants from the Canadian Institutes of Health Research and the United States Muscular Dystrophy Association (to S. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Center for Research in Neuroscience, McGill University, Montréal General Hospital Research Institute, 1650 Cedar, Montréal, Québec, Canada, H3G 1A4. Tel.: 514-934-1934, ext. 44237; Fax: 514-934-8265; E-mail: Sal.carbonetto{at}mcgill.ca.

2 The abbreviations used are: DG, dystroglycan; AChE, acetylcholinesterase; AChR, acetylcholine receptor; CMV, cytomegalovirus; DGC, dystrophin-associated glycoprotein complex; ECM, extracellular matrix; eGFP, enhanced green fluorescent protein; FGF-2, fibroblast growth factor 2; mAb, monoclonal antibody; MuSK, muscle-specific kinase; NMJ, neuromuscular junction; ES, embryonic stem; DPBS, Dulbecco's phosphate-buffered saline. Back

3 H. Peng, J. Vincent-Heroux, and S. Carbonetto, unpublished observations. Back

4 M. R. Tremblay and S. Carbonetto, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank current and past members of the Carbonetto laboratory, especially Christian Jacobson, Patrice Côté, Ramin Raouf, and Huashan Peng for technical support. We also thank Dr. Michael Ferns (University of California, Davis) for the recombinant agrin and Dr. Steve Winder (University of Sheffield, UK) for the DG mutant cDNA constructs. We also acknowledge Prof. Jose Correa (McGill University) for help with the statistical analysis.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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