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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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The distribution of various basement membrane molecules on myotubes derived from wild-type and dystroglycan- ES cells. Wild-type (R1) and dystroglycan- myotubes (3C12) were fixed gently and stained with antibodies for the basement membrane molecules laminin, perlecan, collagen IV, fibronectin, and agrin. Occasionally this fixation permeablizes myotubes, which results in some intracellular labeling that highlights nuclei (A, left corner), though the vast majority of the cells in this and other panels show surface labeling and have no visible intracellular labeling. Photomicrographs were taken of fields of myotubes lacking AChR clusters. Over most of the sarcolemma there appeared to be few morphological differences in basement membranes of wild-type and dystroglycan- myotubes, with the exception of laminin, which was found consistently to form large plaques on wild-type myotubes (arrow in A), but not on dystroglycan- myotubes (A′).
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Figure 10: The distribution of various basement membrane molecules on myotubes derived from wild-type and dystroglycan- ES cells. Wild-type (R1) and dystroglycan- myotubes (3C12) were fixed gently and stained with antibodies for the basement membrane molecules laminin, perlecan, collagen IV, fibronectin, and agrin. Occasionally this fixation permeablizes myotubes, which results in some intracellular labeling that highlights nuclei (A, left corner), though the vast majority of the cells in this and other panels show surface labeling and have no visible intracellular labeling. Photomicrographs were taken of fields of myotubes lacking AChR clusters. Over most of the sarcolemma there appeared to be few morphological differences in basement membranes of wild-type and dystroglycan- myotubes, with the exception of laminin, which was found consistently to form large plaques on wild-type myotubes (arrow in A), but not on dystroglycan- myotubes (A′).

Mentions: Fresh-frozen, superficial gluteal muscles from chimeric mice aged between 3 and 12 wk were sectioned longitudinally (10 μm), blocked with 10% goat serum, and stained with TRITC–α-Btx and affinity-purified antidystroglycan antiserum, followed by an Oregon green 488–conjugated secondary antiserum. Myotubes from differentiated ES cells were incubated at 37°C with agrin (500 pM; a gift from M. Ferns, McGill University; Ferns et al. 1993) or EHS laminin (100 nM; Timpl et al. 1982) overnight in ES cell differentiation medium. In all cases AChRs were visualized by staining cultures of live cells. TRITC- or Alexa 488–labeled α-Btx (2 μg/ml) was added directly to the media for 1 h at 37°C. Cultures were then washed twice with PBS and fixed with 2% paraformaldehyde for 15 min at 37°C. This fixation permeablizes a small fraction of the myotubes and occasionally myotubes will be labeled intracellularly (see Fig. 10 A). After two additional washes with PBS, cells were blocked for 1 h with 10% goat serum in PBS (blocking buffer). When primary antibodies recognizing intracellular epitopes were used, cells were first permeablized with 0.1% Triton X-100 in blocking buffer for 10 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated at room temperature for 1 h. After several washes with PBS, bound primary antibody was detected with TRITC- or Oregon green 488–conjugated secondary antiserum. Cultures were washed a final time before the addition of Immuno Floure Mounting Medium (ICN Biomedicals) and mounted on coverslips. Fluorescence was viewed with a ZEISS Axioskop fluorescence microscope. To document observed differences in the intensity of antibody labeling, experimental and control samples were photographed and reproduced under identical conditions.


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)

The distribution of various basement membrane molecules on myotubes derived from wild-type and dystroglycan- ES cells. Wild-type (R1) and dystroglycan- myotubes (3C12) were fixed gently and stained with antibodies for the basement membrane molecules laminin, perlecan, collagen IV, fibronectin, and agrin. Occasionally this fixation permeablizes myotubes, which results in some intracellular labeling that highlights nuclei (A, left corner), though the vast majority of the cells in this and other panels show surface labeling and have no visible intracellular labeling. Photomicrographs were taken of fields of myotubes lacking AChR clusters. Over most of the sarcolemma there appeared to be few morphological differences in basement membranes of wild-type and dystroglycan- myotubes, with the exception of laminin, which was found consistently to form large plaques on wild-type myotubes (arrow in A), but not on dystroglycan- myotubes (A′).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195998&req=5

Figure 10: The distribution of various basement membrane molecules on myotubes derived from wild-type and dystroglycan- ES cells. Wild-type (R1) and dystroglycan- myotubes (3C12) were fixed gently and stained with antibodies for the basement membrane molecules laminin, perlecan, collagen IV, fibronectin, and agrin. Occasionally this fixation permeablizes myotubes, which results in some intracellular labeling that highlights nuclei (A, left corner), though the vast majority of the cells in this and other panels show surface labeling and have no visible intracellular labeling. Photomicrographs were taken of fields of myotubes lacking AChR clusters. Over most of the sarcolemma there appeared to be few morphological differences in basement membranes of wild-type and dystroglycan- myotubes, with the exception of laminin, which was found consistently to form large plaques on wild-type myotubes (arrow in A), but not on dystroglycan- myotubes (A′).
Mentions: Fresh-frozen, superficial gluteal muscles from chimeric mice aged between 3 and 12 wk were sectioned longitudinally (10 μm), blocked with 10% goat serum, and stained with TRITC–α-Btx and affinity-purified antidystroglycan antiserum, followed by an Oregon green 488–conjugated secondary antiserum. Myotubes from differentiated ES cells were incubated at 37°C with agrin (500 pM; a gift from M. Ferns, McGill University; Ferns et al. 1993) or EHS laminin (100 nM; Timpl et al. 1982) overnight in ES cell differentiation medium. In all cases AChRs were visualized by staining cultures of live cells. TRITC- or Alexa 488–labeled α-Btx (2 μg/ml) was added directly to the media for 1 h at 37°C. Cultures were then washed twice with PBS and fixed with 2% paraformaldehyde for 15 min at 37°C. This fixation permeablizes a small fraction of the myotubes and occasionally myotubes will be labeled intracellularly (see Fig. 10 A). After two additional washes with PBS, cells were blocked for 1 h with 10% goat serum in PBS (blocking buffer). When primary antibodies recognizing intracellular epitopes were used, cells were first permeablized with 0.1% Triton X-100 in blocking buffer for 10 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated at room temperature for 1 h. After several washes with PBS, bound primary antibody was detected with TRITC- or Oregon green 488–conjugated secondary antiserum. Cultures were washed a final time before the addition of Immuno Floure Mounting Medium (ICN Biomedicals) and mounted on coverslips. Fluorescence was viewed with a ZEISS Axioskop fluorescence microscope. To document observed differences in the intensity of antibody labeling, experimental and control samples were photographed and reproduced under identical conditions.

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