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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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Agrin and rapsyn colocalize with AChR clusters on dystroglycan- myotubes. Myotubes from wild-type (R1) and dystroglycan- ES cells (3C12) were treated overnight with 500 pM agrin and stained with TRITC–α-Btx and either antiagrin or antirapsyn antibodies. In both cell types there is colocalization of AChR clusters with agrin (arrows in A, A′, B, and B′) and with rapsyn (arrows in C, C′, D, and D′). The percentage overlap (E and F) indicates the percentage of AChR clusters with corresponding concentrations of agrin and rapsyn. In both 3C12 and R1 lines we found no significant difference in the level of colocalization of AChR aggregates with agrin (E) or rapsyn (F). The histograms represent the average of three separate experiments in which 10–20 microscopic fields containing at least one AChR cluster per field. Bar, 10 μm.
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Figure 5: Agrin and rapsyn colocalize with AChR clusters on dystroglycan- myotubes. Myotubes from wild-type (R1) and dystroglycan- ES cells (3C12) were treated overnight with 500 pM agrin and stained with TRITC–α-Btx and either antiagrin or antirapsyn antibodies. In both cell types there is colocalization of AChR clusters with agrin (arrows in A, A′, B, and B′) and with rapsyn (arrows in C, C′, D, and D′). The percentage overlap (E and F) indicates the percentage of AChR clusters with corresponding concentrations of agrin and rapsyn. In both 3C12 and R1 lines we found no significant difference in the level of colocalization of AChR aggregates with agrin (E) or rapsyn (F). The histograms represent the average of three separate experiments in which 10–20 microscopic fields containing at least one AChR cluster per field. Bar, 10 μm.

Mentions: Agrin binds directly to α-dystroglycan (Bowe et al. 1994; Campanelli et al. 1994; Gee et al. 1994; Sugiyama et al. 1994) and is found in microcluster of AChRs within 1 h of synapse formation (Cohen et al. 1995). In addition, β-dystroglycan has been reported to aggregate with rapsyn and AChRs when coexpressed in heterologous cells and to bind to rapsyn in biochemical assays (Apel et al. 1995; Cartaud et al. 1998). We examined whether the microclusters on dystroglycan- cells were similar in composition to those at nascent synapses and whether they contained agrin. Fixed ES cell myotubes were stained with an antiagrin polyclonal antiserum in conjunction with live staining with TRITC–α-Btx to determine whether agrin codistributed with AChR clusters. We found that agrin was localized to AChR clusters on wild-type (R1) myotubes (Fig. 5A and A′) as well as dystroglycan- (3C12) myotubes (Fig. 5B and Fig. B′). R1 and 3C12 cells had 88.9 ± 4.0% and 81.7 ± 8.0% overlap of agrin labeling with AChR clusters, respectively (Fig. 5 E). Antirapsyn antibodies labeled 91.7 ± 8.3% of R1 AChR clusters (Fig. 5C, Fig. C′, D, and D′) and 75 ± 13.1% of 3C12 clusters (Fig. 5 F). However, despite similar levels of colocalization there were differences in the intensity and distribution of rapsyn staining in 3C12 myotubes. In wild-type myotubes rapsyn was evenly distributed throughout each AChR cluster, but in dystroglycan- myotubes rapsyn was restricted to the largest and brightest regions of all clusters. The presence of agrin and rapsyn in microclusters of AChRs in dystroglycan- cells is consistent with the notion that these are microclusters similar to those at developing NMJs and suggest that they are localized there independent of dystroglycan.


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)

Agrin and rapsyn colocalize with AChR clusters on dystroglycan- myotubes. Myotubes from wild-type (R1) and dystroglycan- ES cells (3C12) were treated overnight with 500 pM agrin and stained with TRITC–α-Btx and either antiagrin or antirapsyn antibodies. In both cell types there is colocalization of AChR clusters with agrin (arrows in A, A′, B, and B′) and with rapsyn (arrows in C, C′, D, and D′). The percentage overlap (E and F) indicates the percentage of AChR clusters with corresponding concentrations of agrin and rapsyn. In both 3C12 and R1 lines we found no significant difference in the level of colocalization of AChR aggregates with agrin (E) or rapsyn (F). The histograms represent the average of three separate experiments in which 10–20 microscopic fields containing at least one AChR cluster per field. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 5: Agrin and rapsyn colocalize with AChR clusters on dystroglycan- myotubes. Myotubes from wild-type (R1) and dystroglycan- ES cells (3C12) were treated overnight with 500 pM agrin and stained with TRITC–α-Btx and either antiagrin or antirapsyn antibodies. In both cell types there is colocalization of AChR clusters with agrin (arrows in A, A′, B, and B′) and with rapsyn (arrows in C, C′, D, and D′). The percentage overlap (E and F) indicates the percentage of AChR clusters with corresponding concentrations of agrin and rapsyn. In both 3C12 and R1 lines we found no significant difference in the level of colocalization of AChR aggregates with agrin (E) or rapsyn (F). The histograms represent the average of three separate experiments in which 10–20 microscopic fields containing at least one AChR cluster per field. Bar, 10 μm.
Mentions: Agrin binds directly to α-dystroglycan (Bowe et al. 1994; Campanelli et al. 1994; Gee et al. 1994; Sugiyama et al. 1994) and is found in microcluster of AChRs within 1 h of synapse formation (Cohen et al. 1995). In addition, β-dystroglycan has been reported to aggregate with rapsyn and AChRs when coexpressed in heterologous cells and to bind to rapsyn in biochemical assays (Apel et al. 1995; Cartaud et al. 1998). We examined whether the microclusters on dystroglycan- cells were similar in composition to those at nascent synapses and whether they contained agrin. Fixed ES cell myotubes were stained with an antiagrin polyclonal antiserum in conjunction with live staining with TRITC–α-Btx to determine whether agrin codistributed with AChR clusters. We found that agrin was localized to AChR clusters on wild-type (R1) myotubes (Fig. 5A and A′) as well as dystroglycan- (3C12) myotubes (Fig. 5B and Fig. B′). R1 and 3C12 cells had 88.9 ± 4.0% and 81.7 ± 8.0% overlap of agrin labeling with AChR clusters, respectively (Fig. 5 E). Antirapsyn antibodies labeled 91.7 ± 8.3% of R1 AChR clusters (Fig. 5C, Fig. C′, D, and D′) and 75 ± 13.1% of 3C12 clusters (Fig. 5 F). However, despite similar levels of colocalization there were differences in the intensity and distribution of rapsyn staining in 3C12 myotubes. In wild-type myotubes rapsyn was evenly distributed throughout each AChR cluster, but in dystroglycan- myotubes rapsyn was restricted to the largest and brightest regions of all clusters. The presence of agrin and rapsyn in microclusters of AChRs in dystroglycan- cells is consistent with the notion that these are microclusters similar to those at developing NMJs and suggest that they are localized there independent of dystroglycan.

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