Limits...
The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane.

Jacobson C, Côté PD, Rossi SG, Rotundo RL, Carbonetto S - J. Cell Biol. (2001)

Bottom Line: The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber.These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan.In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University/Center for Neuroscience Research, Montréal General Hospital Research Institute, Montréal, Québec H3G 1A4, Canada.

ABSTRACT
The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber. Defects in the DAP complex have been linked previously to a variety of muscular dystrophies. Other evidence points to a role for the DAP complex in formation of nerve-muscle synapses. We show that myotubes differentiated from dystroglycan-/- embryonic stem cells are responsive to agrin, but produce acetylcholine receptor (AChR) clusters which are two to three times larger in area, about half as dense, and significantly less stable than those on dystroglycan+/+ myotubes. AChRs at neuromuscular junctions are similarly affected in dystroglycan-deficient chimeric mice and there is a coordinate increase in nerve terminal size at these junctions. In culture and in vivo the absence of dystroglycan disrupts the localization to AChR clusters of laminin, perlecan, and acetylcholinesterase (AChE), but not rapsyn or agrin. Treatment of myotubes in culture with laminin induces AChR clusters on dystroglycan+/+, but not -/- myotubes. These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan. In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

Show MeSH

Related in: MedlinePlus

Laminin colocalization with AChR aggregates is disrupted on dystroglycan- myotubes. Wild-type (R1) and dystroglycan- myotubes (3C12) were treated overnight with 500 pM agrin and subsequently fixed and stained for AChRs in conjunction with either anti–EHS laminin (A, A′, B, and B′) or antilaminin α2 chain (merosin) antibodies (C, C′, D, and D′). The filled arrows in all panels indicate the location of AChR clusters (A–D), laminin (A′ and B′), or merosin (C′ and D′). AChR clusters on the tops of wild-type myotubes contained both laminin and merosin whereas those on dystroglycan- cells have little or no laminin and merosin. We did note some ribbons of AChRs along the sides of myotubes which contain laminin (open arrows). The amount of overlap with AChR clusters for each laminin isoform is shown in E and F. Histograms represent the quantification of three separate experiments with 10–20 microscope fields containing at least one AChR cluster per field. Significance was determined using Student's t test (*P < 0.0001). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195998&req=5

Figure 6: Laminin colocalization with AChR aggregates is disrupted on dystroglycan- myotubes. Wild-type (R1) and dystroglycan- myotubes (3C12) were treated overnight with 500 pM agrin and subsequently fixed and stained for AChRs in conjunction with either anti–EHS laminin (A, A′, B, and B′) or antilaminin α2 chain (merosin) antibodies (C, C′, D, and D′). The filled arrows in all panels indicate the location of AChR clusters (A–D), laminin (A′ and B′), or merosin (C′ and D′). AChR clusters on the tops of wild-type myotubes contained both laminin and merosin whereas those on dystroglycan- cells have little or no laminin and merosin. We did note some ribbons of AChRs along the sides of myotubes which contain laminin (open arrows). The amount of overlap with AChR clusters for each laminin isoform is shown in E and F. Histograms represent the quantification of three separate experiments with 10–20 microscope fields containing at least one AChR cluster per field. Significance was determined using Student's t test (*P < 0.0001). Bar, 10 μm.

Mentions: In view of reports (Montanaro et al. 1998; Peng et al. 1998, Peng et al. 1999) that α-dystroglycan is a receptor for laminin, perlecan, and indirectly AChE, all of which are important components of the synaptic basement membrane, we examined the distribution of these molecules in differentiated ES cell cultures for dystroglycan. We initially stained myotubes with two laminin-specific antibodies. With an antiserum to EHS laminin, staining was found to be highly concentrated at AChR clusters in wild-type myotubes (Fig. 6A and A′), but not in dystroglycan- myotubes (Fig. 6B and Fig. B′). In many cases the size and shape of the of these laminin clusters mirrored that of the underlying AChRs. Laminin colocalized with AChRs on wild-type (92.1 ± 2.6%) and dystroglycan- (16.3 ± 6.0%) myotubes (Fig. 6 E). Similar results were obtained with antiserum specific for the laminin α2 chain (Fig. 6C, Fig. C′, D, and D′): the α chain is part of laminins 2 and 4, which are the predominant isoforms on the muscle surface (Ehrig et al. 1990). When quantified, laminin α2 staining overlapped with 90 ± 4.3% of AChR clusters on R1 myotubes and 20 ± 10.7% on the surface of 3C12 myotubes (Fig. 6 F).


The dystroglycan complex is necessary for stabilization of acetylcholine receptor clusters at neuromuscular junctions and formation of the synaptic basement membrane.

Jacobson C, Côté PD, Rossi SG, Rotundo RL, Carbonetto S - J. Cell Biol. (2001)

Laminin colocalization with AChR aggregates is disrupted on dystroglycan- myotubes. Wild-type (R1) and dystroglycan- myotubes (3C12) were treated overnight with 500 pM agrin and subsequently fixed and stained for AChRs in conjunction with either anti–EHS laminin (A, A′, B, and B′) or antilaminin α2 chain (merosin) antibodies (C, C′, D, and D′). The filled arrows in all panels indicate the location of AChR clusters (A–D), laminin (A′ and B′), or merosin (C′ and D′). AChR clusters on the tops of wild-type myotubes contained both laminin and merosin whereas those on dystroglycan- cells have little or no laminin and merosin. We did note some ribbons of AChRs along the sides of myotubes which contain laminin (open arrows). The amount of overlap with AChR clusters for each laminin isoform is shown in E and F. Histograms represent the quantification of three separate experiments with 10–20 microscope fields containing at least one AChR cluster per field. Significance was determined using Student's t test (*P < 0.0001). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195998&req=5

Figure 6: Laminin colocalization with AChR aggregates is disrupted on dystroglycan- myotubes. Wild-type (R1) and dystroglycan- myotubes (3C12) were treated overnight with 500 pM agrin and subsequently fixed and stained for AChRs in conjunction with either anti–EHS laminin (A, A′, B, and B′) or antilaminin α2 chain (merosin) antibodies (C, C′, D, and D′). The filled arrows in all panels indicate the location of AChR clusters (A–D), laminin (A′ and B′), or merosin (C′ and D′). AChR clusters on the tops of wild-type myotubes contained both laminin and merosin whereas those on dystroglycan- cells have little or no laminin and merosin. We did note some ribbons of AChRs along the sides of myotubes which contain laminin (open arrows). The amount of overlap with AChR clusters for each laminin isoform is shown in E and F. Histograms represent the quantification of three separate experiments with 10–20 microscope fields containing at least one AChR cluster per field. Significance was determined using Student's t test (*P < 0.0001). Bar, 10 μm.
Mentions: In view of reports (Montanaro et al. 1998; Peng et al. 1998, Peng et al. 1999) that α-dystroglycan is a receptor for laminin, perlecan, and indirectly AChE, all of which are important components of the synaptic basement membrane, we examined the distribution of these molecules in differentiated ES cell cultures for dystroglycan. We initially stained myotubes with two laminin-specific antibodies. With an antiserum to EHS laminin, staining was found to be highly concentrated at AChR clusters in wild-type myotubes (Fig. 6A and A′), but not in dystroglycan- myotubes (Fig. 6B and Fig. B′). In many cases the size and shape of the of these laminin clusters mirrored that of the underlying AChRs. Laminin colocalized with AChRs on wild-type (92.1 ± 2.6%) and dystroglycan- (16.3 ± 6.0%) myotubes (Fig. 6 E). Similar results were obtained with antiserum specific for the laminin α2 chain (Fig. 6C, Fig. C′, D, and D′): the α chain is part of laminins 2 and 4, which are the predominant isoforms on the muscle surface (Ehrig et al. 1990). When quantified, laminin α2 staining overlapped with 90 ± 4.3% of AChR clusters on R1 myotubes and 20 ± 10.7% on the surface of 3C12 myotubes (Fig. 6 F).

Bottom Line: The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber.These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan.In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University/Center for Neuroscience Research, Montréal General Hospital Research Institute, Montréal, Québec H3G 1A4, Canada.

ABSTRACT
The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber. Defects in the DAP complex have been linked previously to a variety of muscular dystrophies. Other evidence points to a role for the DAP complex in formation of nerve-muscle synapses. We show that myotubes differentiated from dystroglycan-/- embryonic stem cells are responsive to agrin, but produce acetylcholine receptor (AChR) clusters which are two to three times larger in area, about half as dense, and significantly less stable than those on dystroglycan+/+ myotubes. AChRs at neuromuscular junctions are similarly affected in dystroglycan-deficient chimeric mice and there is a coordinate increase in nerve terminal size at these junctions. In culture and in vivo the absence of dystroglycan disrupts the localization to AChR clusters of laminin, perlecan, and acetylcholinesterase (AChE), but not rapsyn or agrin. Treatment of myotubes in culture with laminin induces AChR clusters on dystroglycan+/+, but not -/- myotubes. These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan. In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.

Show MeSH
Related in: MedlinePlus