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Cell culture-based analysis of postsynaptic membrane assembly in muscle cells.

Teressa G, Prives J - Biol Proced Online (2008)

Bottom Line: We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces.A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ).We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT
We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.

No MeSH data available.


Ef fects on AChR clustering of secondar y plating ofdif ferentiated myotubes onto substrate-coated laminin.Differentiated myotubes replated on laminin-coated coverslips were seen toform increasingly complex and heterogeneous AChR clusters, as visualizedby labeling AChRs on intact myotubes with TMR-Bgt (10nM for 1h at 37°C).The morphological maturation of AChR aggregates proceeds through theovoid stage (2Aa; 2B) on day 1 to pretzel-like aggregates by day 4 postreplating(2Ad; 2B). Scale bar, 20μm.
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Figure 2: Ef fects on AChR clustering of secondar y plating ofdif ferentiated myotubes onto substrate-coated laminin.Differentiated myotubes replated on laminin-coated coverslips were seen toform increasingly complex and heterogeneous AChR clusters, as visualizedby labeling AChRs on intact myotubes with TMR-Bgt (10nM for 1h at 37°C).The morphological maturation of AChR aggregates proceeds through theovoid stage (2Aa; 2B) on day 1 to pretzel-like aggregates by day 4 postreplating(2Ad; 2B). Scale bar, 20μm.

Mentions: Soluble laminin is known to induce ovoid-shaped AChRclusters on muscle cells (5, 6) reminiscent of early stages ofpostsynaptic membrane differentiation upon innervation(3). In contrast, cultured muscle cells plated anddifferentiated in contact with immobilized laminin display far more complex clusters of cell surface AChR resemblingmature postsynaptic membranes of innervated muscle cells (10). We first wanted to use the secondary platingmethod to determine if acute contact with substratelaminin can induce similarly complex AChR clusters onmyotubes. In this method, cultures are allowed todifferentiate into multinucleated myotubes on culturedishes in the absence of exogenous laminin substrate. Thenewly fused myotubes, which express AChR that isdiffusely distributed on the cell surface, are then detachedby mild trypsinization and replated onto laminin-coatedcoverglasses (Fig. 1). Upon contact with the laminincoatedsubstrate, ovoid AChR clusters appeared on theunderside of reattached myotubes within 8-12 hours, as aninitial stage of AChR aggregation (Fig. 2Aa, B).Subsequently, cluster morphology continued to increase incomplexity (Fig. 2) until elaborate pretzel-shaped AChRaggregation structures reminiscent of postsynaptic regionsof innervated muscle were achieved 2-3 days after ovoidclusters first appeared (Fig. 2Ad, B).


Cell culture-based analysis of postsynaptic membrane assembly in muscle cells.

Teressa G, Prives J - Biol Proced Online (2008)

Ef fects on AChR clustering of secondar y plating ofdif ferentiated myotubes onto substrate-coated laminin.Differentiated myotubes replated on laminin-coated coverslips were seen toform increasingly complex and heterogeneous AChR clusters, as visualizedby labeling AChRs on intact myotubes with TMR-Bgt (10nM for 1h at 37°C).The morphological maturation of AChR aggregates proceeds through theovoid stage (2Aa; 2B) on day 1 to pretzel-like aggregates by day 4 postreplating(2Ad; 2B). Scale bar, 20μm.
© Copyright Policy - open acces
Related In: Results  -  Collection

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Figure 2: Ef fects on AChR clustering of secondar y plating ofdif ferentiated myotubes onto substrate-coated laminin.Differentiated myotubes replated on laminin-coated coverslips were seen toform increasingly complex and heterogeneous AChR clusters, as visualizedby labeling AChRs on intact myotubes with TMR-Bgt (10nM for 1h at 37°C).The morphological maturation of AChR aggregates proceeds through theovoid stage (2Aa; 2B) on day 1 to pretzel-like aggregates by day 4 postreplating(2Ad; 2B). Scale bar, 20μm.
Mentions: Soluble laminin is known to induce ovoid-shaped AChRclusters on muscle cells (5, 6) reminiscent of early stages ofpostsynaptic membrane differentiation upon innervation(3). In contrast, cultured muscle cells plated anddifferentiated in contact with immobilized laminin display far more complex clusters of cell surface AChR resemblingmature postsynaptic membranes of innervated muscle cells (10). We first wanted to use the secondary platingmethod to determine if acute contact with substratelaminin can induce similarly complex AChR clusters onmyotubes. In this method, cultures are allowed todifferentiate into multinucleated myotubes on culturedishes in the absence of exogenous laminin substrate. Thenewly fused myotubes, which express AChR that isdiffusely distributed on the cell surface, are then detachedby mild trypsinization and replated onto laminin-coatedcoverglasses (Fig. 1). Upon contact with the laminincoatedsubstrate, ovoid AChR clusters appeared on theunderside of reattached myotubes within 8-12 hours, as aninitial stage of AChR aggregation (Fig. 2Aa, B).Subsequently, cluster morphology continued to increase incomplexity (Fig. 2) until elaborate pretzel-shaped AChRaggregation structures reminiscent of postsynaptic regionsof innervated muscle were achieved 2-3 days after ovoidclusters first appeared (Fig. 2Ad, B).

Bottom Line: We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces.A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ).We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT
We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.

No MeSH data available.