Advertisement

Focal Adhesion Isolation Assay Using ECM-Coated Magnetic Beads

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2217)

Abstract

Focal adhesions are force sensitive structures that dynamically alter their composition, protein-protein interactions, and signaling in response to external mechanical stimuli. These dynamic changes are critical for focal adhesion function and are required for cellular mechanosensing. Here, we describe a simple protocol that allows for isolation of the focal adhesion complex from adherent cells in culture in response to different mechanical stimuli applied at adhesion sites. By combining this assay with approaches such as proteomics or western blot analysis, one can study the force-dependent changes in focal adhesion composition, protein–protein interactions and signaling.

Key words

Focal adhesion composition Magnetics Mechanotransduction 

References

  1. 1.
    Discher DE, Janmey P, Wang YL (2005) Tissue cells feel and respond to the stiffness of their substrate. Science 310(5751):1139–1143.  https://doi.org/10.1126/science.1116995CrossRefPubMedGoogle Scholar
  2. 2.
    Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S (2011) Role of YAP/TAZ in mechanotransduction. Nature 474(7350):179–183.  https://doi.org/10.1038/nature10137CrossRefPubMedGoogle Scholar
  3. 3.
    Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science 302(5651):1704–1709.  https://doi.org/10.1126/science.1092053CrossRefPubMedGoogle Scholar
  4. 4.
    Hoffman BD, Grashoff C, Schwartz MA (2011) Dynamic molecular processes mediate cellular mechanotransduction. Nature 475(7356):316–323.  https://doi.org/10.1038/nature10316CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Humphries JD, Paul NR, Humphries MJ, Morgan MR (2015) Emerging properties of adhesion complexes: what are they and what do they do? Trends Cell Biol 25(7):388–397.  https://doi.org/10.1016/j.tcb.2015.02.008CrossRefPubMedGoogle Scholar
  6. 6.
    Kuo JC, Han X, Hsiao CT, Yates JR 3rd, Waterman CM (2011) Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for beta-Pix in negative regulation of focal adhesion maturation. Nat Cell Biol 13(4):383–393.  https://doi.org/10.1038/ncb2216CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Schiller HB, Fassler R (2013) Mechanosensitivity and compositional dynamics of cell-matrix adhesions. EMBO Rep 14(6):509–519.  https://doi.org/10.1038/embor.2013.49CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Horton ER, Byron A, Askari JA, Ng DHJ, Millon-Fremillon A, Robertson J, Koper EJ, Paul NR, Warwood S, Knight D, Humphries JD, Humphries MJ (2015) Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly. Nat Cell Biol 17(12):1577–1587.  https://doi.org/10.1038/ncb3257CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Byron A Humphries JD Bass MD Knight D Humphries MJ (2011) Proteomic analysis of integrin adhesion complexes. In Science Signaling.  https://doi.org/10.1126/scisignal.2001827
  10. 10.
    Guilluy C, Swaminathan V, Garcia-Mata R, O’Brien ET, Superfine R, Burridge K (2011) The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat Cell Biol 13(6):722–727.  https://doi.org/10.1038/ncb2254CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Marjoram RJ, Guilluy C, Burridge K (2016) Using magnets and magnetic beads to dissect signaling pathways activated by mechanical tension applied to cells. Methods 94:19–26.  https://doi.org/10.1016/j.ymeth.2015.09.025CrossRefPubMedGoogle Scholar
  12. 12.
    Bays JL, Campbell HK, Heidema C, Sebbagh M, DeMali KA (2017) Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK. Nat Cell Biol 19(6):724–731.  https://doi.org/10.1038/ncb3537CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Collins C, Guilluy C, Welch C, O’Brien ET, Hahn K, Superfine R, Burridge K, Tzima E (2012) Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin-RhoA pathway. Curr Biol 22(22):2087–2094.  https://doi.org/10.1016/j.cub.2012.08.051CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Guilluy C, Osborne LD, Van Landeghem L, Sharek L, Superfine R, Garcia-Mata R, Burridge K (2014) Isolated nuclei adapt to force and reveal a mechanotransduction pathway in the nucleus. Nat Cell Biol 16(4):376–381.  https://doi.org/10.1038/ncb2927CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Wallenberg Centre for Molecular Medicine, Division of Oncology, Department of Clinical SciencesLund UniversityLundSweden

Personalised recommendations