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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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AChE distribution is altered at the endplates of dystroglycan-deficient chimeric mouse muscle. Teased muscle fibers from wild-type (A) and chimeric mice (B–D) were isolated and stained with TRITC–α-Btx and fas-2. AChR labeling at endplates varies from nearly normal (B) to severely depleted (C and D). AChE staining is similarly graded and is virtually lost from the most severely disorganized endplates (C′ and D′). The arrows in D and D′ point to punctate aggregates of AChR and the sites to which AChE should be localized within the same field respectively. Bar, 10 μm.
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Figure 8: AChE distribution is altered at the endplates of dystroglycan-deficient chimeric mouse muscle. Teased muscle fibers from wild-type (A) and chimeric mice (B–D) were isolated and stained with TRITC–α-Btx and fas-2. AChR labeling at endplates varies from nearly normal (B) to severely depleted (C and D). AChE staining is similarly graded and is virtually lost from the most severely disorganized endplates (C′ and D′). The arrows in D and D′ point to punctate aggregates of AChR and the sites to which AChE should be localized within the same field respectively. Bar, 10 μm.

Mentions: Perlecan is another extracellular matrix molecule concentrated at the NMJ early during differentiation (Bayne et al. 1984; Anderson 1986). In ES cell myotubes perlecan colocalization with AChRs was dependent on dystroglycan expression. Perlecan was found to be highly colocalized with AChRs in R1 myotubes (Fig. 7A and A′) and the absence of dystroglycan resulted in the loss of perlecan at AChR clusters (Fig. 7B and Fig. B′). Quantitatively, perlecan colocalized with AChRs in 72.9 ± 12% of clusters in wild-type cells and only 10.7 ± 7.7% in myotubes (Fig. 7 E). The loss of perlecan might be expected to affect the distribution of AChE, which can bind to perlecan and is localized to NMJs and AChR clusters (Peng et al. 1999). Wild-type and dystroglycan- myotubes stained with TRITC–α-Btx and Oregon green fas-2 to label AChE revealed AChE at 73.3 ± 11.7% of R1 AChR clusters (Fig. 7C, Fig. C′, and F), but only 8.3 ± 5.6% of 3C12 AChR clusters (Fig. 7D, Fig. D′, and F). We also examined the distribution of AChRs and AChE in teased muscle fibers of dystroglycan-deficient chimeric mice. As shown in Fig. 1, and for reasons explained earlier, we observed a gradation in the disruption of NMJs after labeling with TRITC–α-Btx, reflecting the level of dystroglycan expression at the junction. These defects ranged from very mild (Fig. 8 B), where synaptic gutters or folds in the postsynaptic membrane were abundant and similar in intensity, to wild-type staining at the edges (Fig. 8 A), to very severe (Fig. 8 D) with no distinguishable gutters and punctate AChR labeling. The intensity of fas-2 staining was correlated with the level of disruption of the AChR distribution. When AChR staining was strong, fas-2 staining was intense and its distribution matched that of α-Btx along the synaptic gutters (Fig. 8 B′), as in wild-type muscle (Fig. 8 A′). However, when the AChR distribution was abnormal, fas-2 staining was either weak (Fig. 8 C′) or completely absent (Fig. 8 D′), indicating that AChE localization was dependent on dystroglycan expression at NMJs as at AChR clusters in culture.


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)

AChE distribution is altered at the endplates of dystroglycan-deficient chimeric mouse muscle. Teased muscle fibers from wild-type (A) and chimeric mice (B–D) were isolated and stained with TRITC–α-Btx and fas-2. AChR labeling at endplates varies from nearly normal (B) to severely depleted (C and D). AChE staining is similarly graded and is virtually lost from the most severely disorganized endplates (C′ and D′). The arrows in D and D′ point to punctate aggregates of AChR and the sites to which AChE should be localized within the same field respectively. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: AChE distribution is altered at the endplates of dystroglycan-deficient chimeric mouse muscle. Teased muscle fibers from wild-type (A) and chimeric mice (B–D) were isolated and stained with TRITC–α-Btx and fas-2. AChR labeling at endplates varies from nearly normal (B) to severely depleted (C and D). AChE staining is similarly graded and is virtually lost from the most severely disorganized endplates (C′ and D′). The arrows in D and D′ point to punctate aggregates of AChR and the sites to which AChE should be localized within the same field respectively. Bar, 10 μm.
Mentions: Perlecan is another extracellular matrix molecule concentrated at the NMJ early during differentiation (Bayne et al. 1984; Anderson 1986). In ES cell myotubes perlecan colocalization with AChRs was dependent on dystroglycan expression. Perlecan was found to be highly colocalized with AChRs in R1 myotubes (Fig. 7A and A′) and the absence of dystroglycan resulted in the loss of perlecan at AChR clusters (Fig. 7B and Fig. B′). Quantitatively, perlecan colocalized with AChRs in 72.9 ± 12% of clusters in wild-type cells and only 10.7 ± 7.7% in myotubes (Fig. 7 E). The loss of perlecan might be expected to affect the distribution of AChE, which can bind to perlecan and is localized to NMJs and AChR clusters (Peng et al. 1999). Wild-type and dystroglycan- myotubes stained with TRITC–α-Btx and Oregon green fas-2 to label AChE revealed AChE at 73.3 ± 11.7% of R1 AChR clusters (Fig. 7C, Fig. C′, and F), but only 8.3 ± 5.6% of 3C12 AChR clusters (Fig. 7D, Fig. D′, and F). We also examined the distribution of AChRs and AChE in teased muscle fibers of dystroglycan-deficient chimeric mice. As shown in Fig. 1, and for reasons explained earlier, we observed a gradation in the disruption of NMJs after labeling with TRITC–α-Btx, reflecting the level of dystroglycan expression at the junction. These defects ranged from very mild (Fig. 8 B), where synaptic gutters or folds in the postsynaptic membrane were abundant and similar in intensity, to wild-type staining at the edges (Fig. 8 A), to very severe (Fig. 8 D) with no distinguishable gutters and punctate AChR labeling. The intensity of fas-2 staining was correlated with the level of disruption of the AChR distribution. When AChR staining was strong, fas-2 staining was intense and its distribution matched that of α-Btx along the synaptic gutters (Fig. 8 B′), as in wild-type muscle (Fig. 8 A′). However, when the AChR distribution was abnormal, fas-2 staining was either weak (Fig. 8 C′) or completely absent (Fig. 8 D′), indicating that AChE localization was dependent on dystroglycan expression at NMJs as at AChR clusters in culture.

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