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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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Laminin induces AChR clusters on myotubes derived from wild-type (R1) but not dystroglycan- (3C12) ES cells. Cultures were treated overnight with either 500 pM of agrin or 100 nM EHS laminin, or left untreated (control). The cultures were then stained with α-Btx and the number of AChR aggregates quantified. Agrin and laminin induced a significant increase in the number of AChR clusters on R1 cells but only agrin was effective in inducing clusters on 3C12 myotubes (ANOVA, P < 0.001, n = 3) The values represent the mean ± SD of three individual experiments in which at least 10 microscopic fields per treatment were quantified.
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Figure 11: Laminin induces AChR clusters on myotubes derived from wild-type (R1) but not dystroglycan- (3C12) ES cells. Cultures were treated overnight with either 500 pM of agrin or 100 nM EHS laminin, or left untreated (control). The cultures were then stained with α-Btx and the number of AChR aggregates quantified. Agrin and laminin induced a significant increase in the number of AChR clusters on R1 cells but only agrin was effective in inducing clusters on 3C12 myotubes (ANOVA, P < 0.001, n = 3) The values represent the mean ± SD of three individual experiments in which at least 10 microscopic fields per treatment were quantified.

Mentions: The absence of laminin, but not agrin (compare Fig. 5, top, to Fig. 6), from AChR clusters suggested that laminin, rather than agrin, bound to dystroglycan participates in the condensation of microclusters of AChRs into larger clusters. To test this possibility, we examined the effect of exogenously added laminin on AChR clustering on R1 and 3C12 myotubes. Laminin induced the formation of AChR clusters on the surface of myotubes derived from wild-type ES cells, but not on dystroglycan- myotubes (Fig. 11). Laminin-treated R1 cells had 7.2 vs. 3.2 clusters per field in untreated myotubes, whereas dystroglycan- 3C12 myotubes treated with laminin had 1.5 vs. 2.3 clusters per field in untreated cultures (P < 0.001, n = 3). Laminin-induced AChR clusters on R1 myotubes (Fig. 3, bottom left) were ∼1.7 times larger (17.6 μm2) than those induced by agrin (10.3 μm2; P < 0.0001, n = 50). In comparison, AChR clusters on laminin-treated 3C12 cells (11.4 μm2) were similar in appearance (compare Fig. 3 A, top right with bottom right) and size to spontaneous clusters (13.7 μm2) on untreated cells. The inability of laminin to induce AChR clusters on dystroglycan- myotubes, in combination with the change in appearance of spontaneous and agrin-induced clusters, suggested that laminin–dystroglycan interactions were important in the formation of dense AChR clusters and that disruption of these interactions is responsible for the diffuse, unstable clusters in culture and in vivo.


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 induces AChR clusters on myotubes derived from wild-type (R1) but not dystroglycan- (3C12) ES cells. Cultures were treated overnight with either 500 pM of agrin or 100 nM EHS laminin, or left untreated (control). The cultures were then stained with α-Btx and the number of AChR aggregates quantified. Agrin and laminin induced a significant increase in the number of AChR clusters on R1 cells but only agrin was effective in inducing clusters on 3C12 myotubes (ANOVA, P < 0.001, n = 3) The values represent the mean ± SD of three individual experiments in which at least 10 microscopic fields per treatment were quantified.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 11: Laminin induces AChR clusters on myotubes derived from wild-type (R1) but not dystroglycan- (3C12) ES cells. Cultures were treated overnight with either 500 pM of agrin or 100 nM EHS laminin, or left untreated (control). The cultures were then stained with α-Btx and the number of AChR aggregates quantified. Agrin and laminin induced a significant increase in the number of AChR clusters on R1 cells but only agrin was effective in inducing clusters on 3C12 myotubes (ANOVA, P < 0.001, n = 3) The values represent the mean ± SD of three individual experiments in which at least 10 microscopic fields per treatment were quantified.
Mentions: The absence of laminin, but not agrin (compare Fig. 5, top, to Fig. 6), from AChR clusters suggested that laminin, rather than agrin, bound to dystroglycan participates in the condensation of microclusters of AChRs into larger clusters. To test this possibility, we examined the effect of exogenously added laminin on AChR clustering on R1 and 3C12 myotubes. Laminin induced the formation of AChR clusters on the surface of myotubes derived from wild-type ES cells, but not on dystroglycan- myotubes (Fig. 11). Laminin-treated R1 cells had 7.2 vs. 3.2 clusters per field in untreated myotubes, whereas dystroglycan- 3C12 myotubes treated with laminin had 1.5 vs. 2.3 clusters per field in untreated cultures (P < 0.001, n = 3). Laminin-induced AChR clusters on R1 myotubes (Fig. 3, bottom left) were ∼1.7 times larger (17.6 μm2) than those induced by agrin (10.3 μm2; P < 0.0001, n = 50). In comparison, AChR clusters on laminin-treated 3C12 cells (11.4 μm2) were similar in appearance (compare Fig. 3 A, top right with bottom right) and size to spontaneous clusters (13.7 μm2) on untreated cells. The inability of laminin to induce AChR clusters on dystroglycan- myotubes, in combination with the change in appearance of spontaneous and agrin-induced clusters, suggested that laminin–dystroglycan interactions were important in the formation of dense AChR clusters and that disruption of these interactions is responsible for the diffuse, unstable clusters in culture and in vivo.

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