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

Biological Procedures Online volume 10, pages 58–65 (2008)Cite this article

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

References

  1. Glass DJ et al. Agrin acts via a MuSK receptor complex. Cell 1996; 85:513–23.

    Article  PubMed  CAS  Google Scholar 

  2. Hoch W. Formation of the neuromuscular junction. Agrin and its unusual receptors. Eur J Biochem 1999; 265:1–10.

    Article  PubMed  CAS  Google Scholar 

  3. Sanes JR, Lichtman JW. Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 1999; 22:389–442.

    Article  PubMed  CAS  Google Scholar 

  4. Sanes JR, Lichtman JW. Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nat Rev Neurosci 2001; 2:791–805.

    Article  PubMed  CAS  Google Scholar 

  5. Patton BL et al. Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4. Nat Neurosci 2001; 4:597–604.

    Article  PubMed  CAS  Google Scholar 

  6. Patton BL, Miner JH, Chiu AY, Sanes JR. Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice. J Cell Biol 1997; 139:1507–21.

    Article  PubMed  CAS  Google Scholar 

  7. Sugiyama JE, Glass DJ, Yancopoulos GD, Hall ZW. Laminin-induced acetylcholine receptor clustering: an alternative pathway. J Cell Biol 1997; 139:181–91.

    Article  PubMed  CAS  Google Scholar 

  8. Burden SJ. The formation of neuromuscular synapses. Genes Dev 1998; 12:133–48.

    Article  PubMed  CAS  Google Scholar 

  9. Colledge M, Froehner SC. To muster a cluster: anchoring neurotransmitter receptors at synapses. Proc Natl Acad Sci U S A 1998; 95:3341–3.

    Article  PubMed  CAS  Google Scholar 

  10. Kummer TT, Misgeld T, Lichtman JW, Sanes JR. Nerveindependent formation of a topologically complex postsynaptic apparatus. J Cell Biol 2004; 164:1077–1087.

    Article  PubMed  CAS  Google Scholar 

  11. Foster RF, Thompson JM, Kaufman SJ. A laminin substrate promotes myogenesis in rat skeletal muscle cultures: analysis of replication and development using antidesmin and anti-BrdUrd monoclonal antibodies. Dev Biol 1987; 122:11–20.

    Article  PubMed  CAS  Google Scholar 

  12. Weston C, Gordon C, Teressa G, Hod E, Ren XD, Prives J. Cooperative regulation by Rac and Rho of agrininduced acetylcholine receptor clustering in muscle cells. J Biol Chem 2003; 278:6450–5.

    Article  PubMed  CAS  Google Scholar 

  13. Weston C, Yee B, Hod E, Prives J. Agrin-induced acetylcholine receptor clustering is mediated by the small guanosine triphosphatases Rac and Cdc42. J Cell Biol 2000; 150:205–12.

    Article  PubMed  CAS  Google Scholar 

  14. Weston CA, Teressa G, Weeks BS, Prives J. Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways. J Cell Sci 2007; 120:868–875.

    Article  PubMed  CAS  Google Scholar 

  15. Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J 2000; 348:241–55.

    Article  PubMed  CAS  Google Scholar 

  16. Hall A. Rho GTPases and the actin cytoskeleton. Science 1998; 279:509–14.

    Article  PubMed  CAS  Google Scholar 

  17. Ridley AJ. Rho family proteins: coordinating cell responses. Trends Cell Biol 2001; 11:471–7.

    Article  PubMed  CAS  Google Scholar 

  18. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev 2001; 81:153–208.

    PubMed  CAS  Google Scholar 

  19. Ishizaki T et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 2000; 57:976–83.

    PubMed  CAS  Google Scholar 

  20. Uehata M et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997; 389:990–4.

    Article  PubMed  CAS  Google Scholar 

  21. Burkin DJ, Gu M, Hodges BL, Campanelli JT, Kaufman SJ. A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering. J Cell Biol 1998; 143:1067–75.

    Article  PubMed  CAS  Google Scholar 

  22. Lee LK, Kunkel DD, Stollberg J. Mechanistic distinctions between agrin and laminin-1 induced aggregation of acetylcholine receptors. BMC Neurosci 2002; 3:10.

    Article  PubMed  Google Scholar 

  23. Montanaro F et al. Laminin and alpha-dystroglycan mediate acetylcholine receptor aggregation via a MuSK-independent pathway. J Neurosci 1998; 18:1250–18:1260.

    Google Scholar 

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Authors and Affiliations

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

    Getu Teressa & Joav Prives

  2. Medical Scientist Training Program, Stony Brook University, 11794, Stony Brook, NY, USA

    Getu Teressa

Authors
  1. Getu Teressa
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  2. Joav Prives
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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://doiorg.publicaciones.saludcastillayleon.es/10.1251/bpo143

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1251/bpo143

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Biological Procedures Online

ISSN: 1480-9222

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