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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.


Combinatorial ef fects of agrin and laminin in replatedmyotubes.To study the combined effects of agrin and laminin, replatedmyotubes were used to analyze AChR aggregation patterns induced bysoluble and immobilized agrin and laminin. AChR clusters on myotubesreplated onto substrate coated with laminin are shown in panel b. Agrin hasno effect when immobilized either alone (panel a) or in combination withsubstrate laminin (panel c), while soluble agrin redistributes substratelaminin-induced complex AChR aggregation (panel e) into a pattern morecharacteristic of soluble agrin (panel f). Unlike soluble agrin, soluble laminindoes not have any discernible effect on substrate laminin induced complexAChR cluster formation (panel d). Scale bar, 20μm. Immob=Immobilized;Sol=Soluble.
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Figure 5: Combinatorial ef fects of agrin and laminin in replatedmyotubes.To study the combined effects of agrin and laminin, replatedmyotubes were used to analyze AChR aggregation patterns induced bysoluble and immobilized agrin and laminin. AChR clusters on myotubesreplated onto substrate coated with laminin are shown in panel b. Agrin hasno effect when immobilized either alone (panel a) or in combination withsubstrate laminin (panel c), while soluble agrin redistributes substratelaminin-induced complex AChR aggregation (panel e) into a pattern morecharacteristic of soluble agrin (panel f). Unlike soluble agrin, soluble laminindoes not have any discernible effect on substrate laminin induced complexAChR cluster formation (panel d). Scale bar, 20μm. Immob=Immobilized;Sol=Soluble.

Mentions: We have utilized the myotube replating protocol to studythe combined actions of soluble and immobilized forms ofagrin and laminin on AChR aggregation. We found thatsoluble agrin radically modifies the aggregation pattern ofAChR that is induced by immobilized laminin. Exposureto soluble agrin reorganizes the complex clusters into asimpler, ovoid morphology characteristic of agrin clustersas well as their subsequent displacement to the myotubeperiphery by 2 days after the initial exposure to agrin(Fig 5e). Surprisingly, these effects are manifested even under conditions where the exposure duration of replatedmyotubes to agrin is as brief as 15min.


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

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

Combinatorial ef fects of agrin and laminin in replatedmyotubes.To study the combined effects of agrin and laminin, replatedmyotubes were used to analyze AChR aggregation patterns induced bysoluble and immobilized agrin and laminin. AChR clusters on myotubesreplated onto substrate coated with laminin are shown in panel b. Agrin hasno effect when immobilized either alone (panel a) or in combination withsubstrate laminin (panel c), while soluble agrin redistributes substratelaminin-induced complex AChR aggregation (panel e) into a pattern morecharacteristic of soluble agrin (panel f). Unlike soluble agrin, soluble laminindoes not have any discernible effect on substrate laminin induced complexAChR cluster formation (panel d). Scale bar, 20μm. Immob=Immobilized;Sol=Soluble.
© Copyright Policy - open acces
Related In: Results  -  Collection

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Figure 5: Combinatorial ef fects of agrin and laminin in replatedmyotubes.To study the combined effects of agrin and laminin, replatedmyotubes were used to analyze AChR aggregation patterns induced bysoluble and immobilized agrin and laminin. AChR clusters on myotubesreplated onto substrate coated with laminin are shown in panel b. Agrin hasno effect when immobilized either alone (panel a) or in combination withsubstrate laminin (panel c), while soluble agrin redistributes substratelaminin-induced complex AChR aggregation (panel e) into a pattern morecharacteristic of soluble agrin (panel f). Unlike soluble agrin, soluble laminindoes not have any discernible effect on substrate laminin induced complexAChR cluster formation (panel d). Scale bar, 20μm. Immob=Immobilized;Sol=Soluble.
Mentions: We have utilized the myotube replating protocol to studythe combined actions of soluble and immobilized forms ofagrin and laminin on AChR aggregation. We found thatsoluble agrin radically modifies the aggregation pattern ofAChR that is induced by immobilized laminin. Exposureto soluble agrin reorganizes the complex clusters into asimpler, ovoid morphology characteristic of agrin clustersas well as their subsequent displacement to the myotubeperiphery by 2 days after the initial exposure to agrin(Fig 5e). Surprisingly, these effects are manifested even under conditions where the exposure duration of replatedmyotubes to agrin is as brief as 15min.

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.