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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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AChR cluster stability is reduced in dystroglycan- myotubes. Differentiated ES cells were incubated overnight with or without 500 pM agrin, washed, then maintained in fresh medium. Cultures were labeled with TRITC–α-Btx at 0, 4, and 8 h after washing, fixed, and the number of AChR clusters per field was determined. The number of spontaneous clusters in untreated cultures was subtracted from each of the agrin treated time points and the resulting value was expressed as a percentage of the value at time zero. AChR clusters on 3C12 cells dispersed at approximately three times the rate of clusters on wild-type cells. By 8 h there was an 88% drop in the number of AChR aggregates on 3C12 cells (filled circles), compared with a 27% drop on R1 cells (open circles). There was no significant difference between the number of clusters remaining on 3C12 cells at 8 h and on untreated cultures. The data represent the average of three trials. Significance was tested by ANOVA (P < 0.0001).
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Figure 4: AChR cluster stability is reduced in dystroglycan- myotubes. Differentiated ES cells were incubated overnight with or without 500 pM agrin, washed, then maintained in fresh medium. Cultures were labeled with TRITC–α-Btx at 0, 4, and 8 h after washing, fixed, and the number of AChR clusters per field was determined. The number of spontaneous clusters in untreated cultures was subtracted from each of the agrin treated time points and the resulting value was expressed as a percentage of the value at time zero. AChR clusters on 3C12 cells dispersed at approximately three times the rate of clusters on wild-type cells. By 8 h there was an 88% drop in the number of AChR aggregates on 3C12 cells (filled circles), compared with a 27% drop on R1 cells (open circles). There was no significant difference between the number of clusters remaining on 3C12 cells at 8 h and on untreated cultures. The data represent the average of three trials. Significance was tested by ANOVA (P < 0.0001).

Mentions: In view of its position as a transmembrane complex and its role in condensing AChRs, it is reasonable to propose that α- and β-dystroglycan contribute to the stabilization of AChR clusters once formed. Cluster stability was determined by quantifying the number of AChR clusters on wild-type and dystroglycan- myotubes after treatment overnight with 500 pM agrin, then replacing the agrin-containing medium with fresh medium and assaying the number of AChR clusters after 0, 4, or 8 h. Within 4 h there was a significant difference in the number of AChR clusters on R1- and 3C12-derived myotubes (Fig. 4). Clusters on R1 cells had decreased by 11%, but on 3C12 cells by 62%. This downward trend continued, with R1 myotubes declining a further 16% and 3C12 myotubes 26% by 8 h (Fig. 4). Indeed, by 8 h, the vast majority of agrin-induced AChR clusters remained on R1 cells, whereas they had essentially dispersed on 3C12 cells. These data suggest that condensation of AChR clusters mediated by dystroglycan enhanced their stability.


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)

AChR cluster stability is reduced in dystroglycan- myotubes. Differentiated ES cells were incubated overnight with or without 500 pM agrin, washed, then maintained in fresh medium. Cultures were labeled with TRITC–α-Btx at 0, 4, and 8 h after washing, fixed, and the number of AChR clusters per field was determined. The number of spontaneous clusters in untreated cultures was subtracted from each of the agrin treated time points and the resulting value was expressed as a percentage of the value at time zero. AChR clusters on 3C12 cells dispersed at approximately three times the rate of clusters on wild-type cells. By 8 h there was an 88% drop in the number of AChR aggregates on 3C12 cells (filled circles), compared with a 27% drop on R1 cells (open circles). There was no significant difference between the number of clusters remaining on 3C12 cells at 8 h and on untreated cultures. The data represent the average of three trials. Significance was tested by ANOVA (P < 0.0001).
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Related In: Results  -  Collection

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Figure 4: AChR cluster stability is reduced in dystroglycan- myotubes. Differentiated ES cells were incubated overnight with or without 500 pM agrin, washed, then maintained in fresh medium. Cultures were labeled with TRITC–α-Btx at 0, 4, and 8 h after washing, fixed, and the number of AChR clusters per field was determined. The number of spontaneous clusters in untreated cultures was subtracted from each of the agrin treated time points and the resulting value was expressed as a percentage of the value at time zero. AChR clusters on 3C12 cells dispersed at approximately three times the rate of clusters on wild-type cells. By 8 h there was an 88% drop in the number of AChR aggregates on 3C12 cells (filled circles), compared with a 27% drop on R1 cells (open circles). There was no significant difference between the number of clusters remaining on 3C12 cells at 8 h and on untreated cultures. The data represent the average of three trials. Significance was tested by ANOVA (P < 0.0001).
Mentions: In view of its position as a transmembrane complex and its role in condensing AChRs, it is reasonable to propose that α- and β-dystroglycan contribute to the stabilization of AChR clusters once formed. Cluster stability was determined by quantifying the number of AChR clusters on wild-type and dystroglycan- myotubes after treatment overnight with 500 pM agrin, then replacing the agrin-containing medium with fresh medium and assaying the number of AChR clusters after 0, 4, or 8 h. Within 4 h there was a significant difference in the number of AChR clusters on R1- and 3C12-derived myotubes (Fig. 4). Clusters on R1 cells had decreased by 11%, but on 3C12 cells by 62%. This downward trend continued, with R1 myotubes declining a further 16% and 3C12 myotubes 26% by 8 h (Fig. 4). Indeed, by 8 h, the vast majority of agrin-induced AChR clusters remained on R1 cells, whereas they had essentially dispersed on 3C12 cells. These data suggest that condensation of AChR clusters mediated by dystroglycan enhanced their stability.

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