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

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.

Abbreviations

AChR:

acetylcholine receptor

EGFP:

enhanced green fluorescent protein

NMJ:

neuromuscular junction

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Correspondence to Joav Prives.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Teressa, G., Prives, J. Cell culture-based analysis of postsynaptic membrane assembly in muscle cells. Biol. Proced. Online 10, 58–65 (2008). https://doi.org/10.1251/bpo143

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  • DOI: https://doi.org/10.1251/bpo143

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