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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 area and density is altered in dystroglycan- myotubes. (A) The histograms show size distributions for the wild-type (R1) and dystroglycan- (3C12) cells lines in untreated cultures (Control) or in cultures treated with 500 pM agrin or 100 nM laminin. The average cluster area is represented by the peak of the dashed line on each graph. Agrin and laminin treatment of R1 cells resulted in an increase in cluster area from 6.9 to 10.3 and 17.6 μm2, respectively. A larger increase in AChR cluster size was noted for agrin-treated 3C12 cells, 31.4 μm2, up from 13.7 μm2. Laminin increased the size of clusters on R1 cells but was ineffective on 3C12 cells. At least 10 microscope fields containing AChR clusters were quantified in five separate experiments for each cell line and treatment. (B) The density of AChR clusters was estimated (see Materials and Methods) for the three cell lines after agrin treatment. AChR clusters on 3C12 and 3H1 cells were twice as diffuse as those on R1 cells. AChRs covered 28 and 30% of the area within clusters on dystroglycan- myotubes versus 63% of the area on wild-type clusters. At least 50 clusters were quantified per cell line over five experiments to determine AChR density and the significance tested using a Student's t test (P < 0.0001).
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Figure 3: AChR cluster area and density is altered in dystroglycan- myotubes. (A) The histograms show size distributions for the wild-type (R1) and dystroglycan- (3C12) cells lines in untreated cultures (Control) or in cultures treated with 500 pM agrin or 100 nM laminin. The average cluster area is represented by the peak of the dashed line on each graph. Agrin and laminin treatment of R1 cells resulted in an increase in cluster area from 6.9 to 10.3 and 17.6 μm2, respectively. A larger increase in AChR cluster size was noted for agrin-treated 3C12 cells, 31.4 μm2, up from 13.7 μm2. Laminin increased the size of clusters on R1 cells but was ineffective on 3C12 cells. At least 10 microscope fields containing AChR clusters were quantified in five separate experiments for each cell line and treatment. (B) The density of AChR clusters was estimated (see Materials and Methods) for the three cell lines after agrin treatment. AChR clusters on 3C12 and 3H1 cells were twice as diffuse as those on R1 cells. AChRs covered 28 and 30% of the area within clusters on dystroglycan- myotubes versus 63% of the area on wild-type clusters. At least 50 clusters were quantified per cell line over five experiments to determine AChR density and the significance tested using a Student's t test (P < 0.0001).

Mentions: Detailed morphometric analysis allowed quantification of the differences in size and density of AChR clusters on R1 and 3C12 myotubes. Cluster area was determined for untreated and agrin-treated R1 and 3C12 myotubes, and the resulting values were used to calculate the average cluster size as well as to generate a frequency distribution of cluster areas (Fig. 3 A). Agrin treatment of wild-type (R1) and (3C12) myotubes resulted in a much larger increase in AChR cluster area in 3C12 cells (13.69 to 31.35 μm2) than in R1 cells (6.93 to 10.3 μm2; Fig. 3 A, broken line). The increase in area with agrin treatment within a cell line and the difference in areas between the two different lines were statistically significant (P < 0.0001). The difference in AChR density was quantified by determining the ratio of surface area covered with α-Btx fluorescence to nonfluorescent area within the perimeter occupied by individual R1 and 3C12 AChR clusters. AChRs occupied ∼63% of the area within the perimeter of a wild-type AChR cluster (Fig. 3 B), which was approximately double the 28–30% of the area that receptors occupied in each dystroglycan- myotube cluster (P < 0.0001; n > 50). The relative level of AChR expression on each ES cell line was estimated by multiplying the number of clusters within each microscope field (Fig. 2) by the average cluster area for each cell type (Fig. 3 A) and the density of AChRs (see above). By this measure the area covered by AChRs on R1, 3C12,and 3H1 myotubes was 78.71, 70.22, and 89.73 μm2, respectively. When expressed relative to values on R1 cells, the values on myotubes were approximately comparable, being 89% (3C12) or 114% (3H1) of the levels on R1 cells. The lack of dystroglycan thus resulted in AChR clusters which were three times larger and twice as diffuse as those found on wild-type cells, though the myotubes appeared to contain similar numbers of AChRs. Taken together, these data suggest that dystroglycan- cells were able to respond to agrin initially to form microclusters but unable to condense them into larger, synaptic-like clusters.


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 area and density is altered in dystroglycan- myotubes. (A) The histograms show size distributions for the wild-type (R1) and dystroglycan- (3C12) cells lines in untreated cultures (Control) or in cultures treated with 500 pM agrin or 100 nM laminin. The average cluster area is represented by the peak of the dashed line on each graph. Agrin and laminin treatment of R1 cells resulted in an increase in cluster area from 6.9 to 10.3 and 17.6 μm2, respectively. A larger increase in AChR cluster size was noted for agrin-treated 3C12 cells, 31.4 μm2, up from 13.7 μm2. Laminin increased the size of clusters on R1 cells but was ineffective on 3C12 cells. At least 10 microscope fields containing AChR clusters were quantified in five separate experiments for each cell line and treatment. (B) The density of AChR clusters was estimated (see Materials and Methods) for the three cell lines after agrin treatment. AChR clusters on 3C12 and 3H1 cells were twice as diffuse as those on R1 cells. AChRs covered 28 and 30% of the area within clusters on dystroglycan- myotubes versus 63% of the area on wild-type clusters. At least 50 clusters were quantified per cell line over five experiments to determine AChR density and the significance tested using a Student's t test (P < 0.0001).
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Related In: Results  -  Collection

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Figure 3: AChR cluster area and density is altered in dystroglycan- myotubes. (A) The histograms show size distributions for the wild-type (R1) and dystroglycan- (3C12) cells lines in untreated cultures (Control) or in cultures treated with 500 pM agrin or 100 nM laminin. The average cluster area is represented by the peak of the dashed line on each graph. Agrin and laminin treatment of R1 cells resulted in an increase in cluster area from 6.9 to 10.3 and 17.6 μm2, respectively. A larger increase in AChR cluster size was noted for agrin-treated 3C12 cells, 31.4 μm2, up from 13.7 μm2. Laminin increased the size of clusters on R1 cells but was ineffective on 3C12 cells. At least 10 microscope fields containing AChR clusters were quantified in five separate experiments for each cell line and treatment. (B) The density of AChR clusters was estimated (see Materials and Methods) for the three cell lines after agrin treatment. AChR clusters on 3C12 and 3H1 cells were twice as diffuse as those on R1 cells. AChRs covered 28 and 30% of the area within clusters on dystroglycan- myotubes versus 63% of the area on wild-type clusters. At least 50 clusters were quantified per cell line over five experiments to determine AChR density and the significance tested using a Student's t test (P < 0.0001).
Mentions: Detailed morphometric analysis allowed quantification of the differences in size and density of AChR clusters on R1 and 3C12 myotubes. Cluster area was determined for untreated and agrin-treated R1 and 3C12 myotubes, and the resulting values were used to calculate the average cluster size as well as to generate a frequency distribution of cluster areas (Fig. 3 A). Agrin treatment of wild-type (R1) and (3C12) myotubes resulted in a much larger increase in AChR cluster area in 3C12 cells (13.69 to 31.35 μm2) than in R1 cells (6.93 to 10.3 μm2; Fig. 3 A, broken line). The increase in area with agrin treatment within a cell line and the difference in areas between the two different lines were statistically significant (P < 0.0001). The difference in AChR density was quantified by determining the ratio of surface area covered with α-Btx fluorescence to nonfluorescent area within the perimeter occupied by individual R1 and 3C12 AChR clusters. AChRs occupied ∼63% of the area within the perimeter of a wild-type AChR cluster (Fig. 3 B), which was approximately double the 28–30% of the area that receptors occupied in each dystroglycan- myotube cluster (P < 0.0001; n > 50). The relative level of AChR expression on each ES cell line was estimated by multiplying the number of clusters within each microscope field (Fig. 2) by the average cluster area for each cell type (Fig. 3 A) and the density of AChRs (see above). By this measure the area covered by AChRs on R1, 3C12,and 3H1 myotubes was 78.71, 70.22, and 89.73 μm2, respectively. When expressed relative to values on R1 cells, the values on myotubes were approximately comparable, being 89% (3C12) or 114% (3H1) of the levels on R1 cells. The lack of dystroglycan thus resulted in AChR clusters which were three times larger and twice as diffuse as those found on wild-type cells, though the myotubes appeared to contain similar numbers of AChRs. Taken together, these data suggest that dystroglycan- cells were able to respond to agrin initially to form microclusters but unable to condense them into larger, synaptic-like clusters.

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