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.
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References
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.1116995
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/nature10137
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.1092053
Hoffman BD, Grashoff C, Schwartz MA (2011) Dynamic molecular processes mediate cellular mechanotransduction. Nature 475(7356):316–323. https://doi.org/10.1038/nature10316
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.2015.02.008
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/ncb2216
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.2013.49
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/ncb3257
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
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/ncb2254
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.2015.09.025
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/ncb3537
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.2012.08.051
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/ncb2927
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Sturgess, W., Swaminathan, V. (2021). Focal Adhesion Isolation Assay Using ECM-Coated Magnetic Beads. In: Vicente-Manzanares, M. (eds) The Integrin Interactome. Methods in Molecular Biology, vol 2217. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0962-0_4
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DOI: https://doi.org/10.1007/978-1-0716-0962-0_4
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Print ISBN: 978-1-0716-0961-3
Online ISBN: 978-1-0716-0962-0
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