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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.

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Adult NMJs are disorganized in dystroglycan-deficient muscles. (A) The specificity of the polyclonal antiserum used in this study against β-dystroglycan was tested by immunoblot of total muscle extracts of wild-type (wt) and dystroglycan-depleted chimeric muscle (ch). Additionally, wild-type muscle sections were probed immunocytochemically using either antiserum against β-dystroglycan (B) or the antiserum preincubated with 100-fold excess of the peptide used for immunization (C and D; phase corresponding to C). Longitudinal sections of the superficial gluteal muscle were stained with TRITC–α-Btx and antisera to β-dystroglycan (anti-βDG). In muscle fibers with near normal levels of dystroglycan expression (E′) there is a concentration of dystroglycan at endplates in a pattern corresponding to AChR staining (E) and equivalent to that seen in wild-type mice (not shown). In muscle fibers with no detectable extrajunctional dystroglycan there may be some dystroglycan at end-plates (F′) and significantly disorganized AChRs (F). In instances where there is no dystroglycan whatsoever (G′), AChRs are grossly disorganized and broken up into microclusters spread over the fiber surface (G). Bar: (B, C, and D) 100 μm; (E, F, and G) 30 μm.
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Figure 1: Adult NMJs are disorganized in dystroglycan-deficient muscles. (A) The specificity of the polyclonal antiserum used in this study against β-dystroglycan was tested by immunoblot of total muscle extracts of wild-type (wt) and dystroglycan-depleted chimeric muscle (ch). Additionally, wild-type muscle sections were probed immunocytochemically using either antiserum against β-dystroglycan (B) or the antiserum preincubated with 100-fold excess of the peptide used for immunization (C and D; phase corresponding to C). Longitudinal sections of the superficial gluteal muscle were stained with TRITC–α-Btx and antisera to β-dystroglycan (anti-βDG). In muscle fibers with near normal levels of dystroglycan expression (E′) there is a concentration of dystroglycan at endplates in a pattern corresponding to AChR staining (E) and equivalent to that seen in wild-type mice (not shown). In muscle fibers with no detectable extrajunctional dystroglycan there may be some dystroglycan at end-plates (F′) and significantly disorganized AChRs (F). In instances where there is no dystroglycan whatsoever (G′), AChRs are grossly disorganized and broken up into microclusters spread over the fiber surface (G). Bar: (B, C, and D) 100 μm; (E, F, and G) 30 μm.

Mentions: In a previous report we described the dystrophic phenotype of chimeric mice deficient in dystroglycan and noted that NMJs, especially in older mice, were disorganized (Côté et al. 1999). At that time we were unable to rigorously evaluate the amount of dystroglycan expressed at individual endplates because high levels of endogenous mouse immunoglobulins were present at the periphery of wild-type muscle fibers and within degenerating fibers of chimeric muscle (Côté et al. 1999). This resulted in a very high level of labeling with the anti–mouse secondary antisera used against the available monoclonal antibody to β-dystroglycan. Thus, in chimeric skeletal muscle, where each myofiber has hundreds to thousands of nuclei, muscles which appeared to be completely derived from dystroglycan- stem cells, when assayed by the extremely sensitive method of glucose phosphate isomerase electrophoresis (De Lorenzo and Ruddle 1969), may have had some residual dystroglycan. To evaluate this, we have generated and characterized a rabbit antiserum against β-dystroglycan. The specificity of this antibody is shown in an immunoblot (Fig. 1 A), where the antiserum identifies a single major band at 43 kD, the size of β-dystroglycan, in wild-type muscle (Fig. 1 A; lane wt). This band is undetectable in muscle extracts from dystroglycan-deficient chimeras (Fig. 1 A; lane ch). Also, immunostaining of wild-type muscles with this antiserum is restricted to the surfaces of these myofibers (Fig. 1 B) and is blocked by addition of the peptide used to generate the antiserum (Fig. 1C and Fig. D). Dystroglycan expression was then assayed by immunohistochemistry on similar muscle sections in conjunction with fluorescently labeled α-Btx to identify NMJs. When viewed en face, we observed endplates on muscle fibers with variable levels of dystroglycan expression. In many cases, in younger mice these junctions appeared like those in muscles from wild-type littermates (Fig. 1E and Fig. E′). In other cases, there was a large reduction of dystroglycan extrasynaptically and the residual dystroglycan had been recruited to partially disorganized endplates (Fig. 1F and Fig. F′); in still other cases, there were fibers devoid of any detectable dystroglycan with very diffuse clusters of AChRs (Fig. 1G and Fig. G′; arrows).


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)

Adult NMJs are disorganized in dystroglycan-deficient muscles. (A) The specificity of the polyclonal antiserum used in this study against β-dystroglycan was tested by immunoblot of total muscle extracts of wild-type (wt) and dystroglycan-depleted chimeric muscle (ch). Additionally, wild-type muscle sections were probed immunocytochemically using either antiserum against β-dystroglycan (B) or the antiserum preincubated with 100-fold excess of the peptide used for immunization (C and D; phase corresponding to C). Longitudinal sections of the superficial gluteal muscle were stained with TRITC–α-Btx and antisera to β-dystroglycan (anti-βDG). In muscle fibers with near normal levels of dystroglycan expression (E′) there is a concentration of dystroglycan at endplates in a pattern corresponding to AChR staining (E) and equivalent to that seen in wild-type mice (not shown). In muscle fibers with no detectable extrajunctional dystroglycan there may be some dystroglycan at end-plates (F′) and significantly disorganized AChRs (F). In instances where there is no dystroglycan whatsoever (G′), AChRs are grossly disorganized and broken up into microclusters spread over the fiber surface (G). Bar: (B, C, and D) 100 μm; (E, F, and G) 30 μm.
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Related In: Results  -  Collection

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Figure 1: Adult NMJs are disorganized in dystroglycan-deficient muscles. (A) The specificity of the polyclonal antiserum used in this study against β-dystroglycan was tested by immunoblot of total muscle extracts of wild-type (wt) and dystroglycan-depleted chimeric muscle (ch). Additionally, wild-type muscle sections were probed immunocytochemically using either antiserum against β-dystroglycan (B) or the antiserum preincubated with 100-fold excess of the peptide used for immunization (C and D; phase corresponding to C). Longitudinal sections of the superficial gluteal muscle were stained with TRITC–α-Btx and antisera to β-dystroglycan (anti-βDG). In muscle fibers with near normal levels of dystroglycan expression (E′) there is a concentration of dystroglycan at endplates in a pattern corresponding to AChR staining (E) and equivalent to that seen in wild-type mice (not shown). In muscle fibers with no detectable extrajunctional dystroglycan there may be some dystroglycan at end-plates (F′) and significantly disorganized AChRs (F). In instances where there is no dystroglycan whatsoever (G′), AChRs are grossly disorganized and broken up into microclusters spread over the fiber surface (G). Bar: (B, C, and D) 100 μm; (E, F, and G) 30 μm.
Mentions: In a previous report we described the dystrophic phenotype of chimeric mice deficient in dystroglycan and noted that NMJs, especially in older mice, were disorganized (Côté et al. 1999). At that time we were unable to rigorously evaluate the amount of dystroglycan expressed at individual endplates because high levels of endogenous mouse immunoglobulins were present at the periphery of wild-type muscle fibers and within degenerating fibers of chimeric muscle (Côté et al. 1999). This resulted in a very high level of labeling with the anti–mouse secondary antisera used against the available monoclonal antibody to β-dystroglycan. Thus, in chimeric skeletal muscle, where each myofiber has hundreds to thousands of nuclei, muscles which appeared to be completely derived from dystroglycan- stem cells, when assayed by the extremely sensitive method of glucose phosphate isomerase electrophoresis (De Lorenzo and Ruddle 1969), may have had some residual dystroglycan. To evaluate this, we have generated and characterized a rabbit antiserum against β-dystroglycan. The specificity of this antibody is shown in an immunoblot (Fig. 1 A), where the antiserum identifies a single major band at 43 kD, the size of β-dystroglycan, in wild-type muscle (Fig. 1 A; lane wt). This band is undetectable in muscle extracts from dystroglycan-deficient chimeras (Fig. 1 A; lane ch). Also, immunostaining of wild-type muscles with this antiserum is restricted to the surfaces of these myofibers (Fig. 1 B) and is blocked by addition of the peptide used to generate the antiserum (Fig. 1C and Fig. D). Dystroglycan expression was then assayed by immunohistochemistry on similar muscle sections in conjunction with fluorescently labeled α-Btx to identify NMJs. When viewed en face, we observed endplates on muscle fibers with variable levels of dystroglycan expression. In many cases, in younger mice these junctions appeared like those in muscles from wild-type littermates (Fig. 1E and Fig. E′). In other cases, there was a large reduction of dystroglycan extrasynaptically and the residual dystroglycan had been recruited to partially disorganized endplates (Fig. 1F and Fig. F′); in still other cases, there were fibers devoid of any detectable dystroglycan with very diffuse clusters of AChRs (Fig. 1G and Fig. G′; arrows).

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