ERK Signaling pp 223-234 | Cite as

Quantifying Tensile Force and ERK Phosphorylation on Actin Stress Fibers

  • Hiroaki HirataEmail author
  • Mukund Gupta
  • Sri Ram Krishna Vedula
  • Chwee Teck Lim
  • Benoit Ladoux
  • Masahiro Sokabe
Part of the Methods in Molecular Biology book series (MIMB, volume 1487)


ERK associates with the actin cytoskeleton, and the actin-associated pool of ERK can be activated (phosphorylated in the activation loop) to induce specific cell responses. Increasing evidence has shown that mechanical conditions of cells significantly affect ERK activation. In particular, tension developed in the actin cytoskeleton has been implicated as a critical mechanism driving ERK signaling. However, a quantitative study of the relationship between actin tension and ERK phosphorylation is missing. In this chapter, we describe our novel methods to quantify tensile force and ERK phosphorylation on individual actin stress fibers. These methods have enabled us to show that ERK is activated on stress fibers in a tensile force-dependent manner.

Key words

Actomyosin Contractility MAP kinase Mechanical stretch Mechanotransduction Micropillar Tension 



This work was supported by the Seed Fund from the Mechanobiology Institute at the National University of Singapore.


  1. 1.
    Ramos JW (2008) The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells. Int J Biochem Cell Biol 40:2707–2719CrossRefPubMedGoogle Scholar
  2. 2.
    Helfman DM, Pawlak G (2005) Myosin light chain kinase and acto-myosin contractility modulate activation of the ERK cascade downstream of oncogenic Ras. J Cell Biochem 95:1069–1080CrossRefPubMedGoogle Scholar
  3. 3.
    Paszek MJ, Zahir N, Johnson KR et al (2005) Tension homeostasis and the malignant phenotype. Cancer Cell 8:241–254CrossRefPubMedGoogle Scholar
  4. 4.
    Sadoshima J, Izumo S (1993) Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J 12:1681–1692PubMedPubMedCentralGoogle Scholar
  5. 5.
    Sawada Y, Nakamura K, Doi K et al (2001) Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase. J Cell Sci 114:1221–1227PubMedGoogle Scholar
  6. 6.
    Assoian RK, Klein EA (2008) Growth control by intracellular tension and extracellular stiffness. Trends Cell Biol 18:347–352CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Numaguchi K, Eguchi S, Yamakawa T et al (1999) Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments. Circ Res 85:5–11CrossRefPubMedGoogle Scholar
  8. 8.
    Vetterkind S, Poythress RH, Lin QQ et al (2013) Hierarchical scaffolding of an ERK1/2 activation pathway. Cell Commun Signal 11:65CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hirata H, Gupta M, Vedula SRK et al (2015) Actomyosin bundles serve as a tension sensor and a platform for ERK activation. EMBO Rep 16:250–257CrossRefPubMedGoogle Scholar
  10. 10.
    Conrad PA, Nederlof MA, Herman IM et al (1989) Correlated distribution of actin, myosin, and microtubules at the leading edge of migrating Swiss 3T3 fibroblasts. Cell Motil Cytoskeleton 14:527–543CrossRefPubMedGoogle Scholar
  11. 11.
    Walston T, Hardin J (2011) Visualizing cell contacts and cell polarity in Caenorhabditis elegans embryos. In: Sharpe J, Wong R (eds) Imaging in developmental biology: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  12. 12.
    Gupta M, Kocgozlu L, Sarangi BR et al (2015) Micropillar substrates: a tool for studying cell mechanobiology. Methods Cell Biol 125:289–308CrossRefPubMedGoogle Scholar
  13. 13.
    Hirata H, Tatsumi H, Sokabe M (2008) Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner. J Cell Sci 121:2795–2804CrossRefPubMedGoogle Scholar
  14. 14.
    Hirata H, Tatsumi H, Lim CT et al (2014) Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions. Am J Physiol Cell Physiol 306:C607–C620CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Hiroaki Hirata
    • 1
    • 2
    Email author
  • Mukund Gupta
    • 1
  • Sri Ram Krishna Vedula
    • 1
    • 3
  • Chwee Teck Lim
    • 1
    • 4
    • 5
  • Benoit Ladoux
    • 1
    • 6
    • 7
  • Masahiro Sokabe
    • 1
    • 8
  1. 1.Mechanobiology InstituteNational University of SingaporeSingaporeSingapore
  2. 2.R-Pharm Japan and Mechanobiology LaboratoryNagoya University Graduate School of MedicineNagoyaJapan
  3. 3.L’oreal Research and InnovationSingaporeSingapore
  4. 4.Department of Biomedical EngineeringNational University of SingaporeSingaporeSingapore
  5. 5.Department of Mechanical EngineeringNational University of SingaporeSingaporeSingapore
  6. 6.Institut Jacques Monod (IJM), CNRS UMR 7592ParisFrance
  7. 7.Université Paris DiderotParisFrance
  8. 8.Mechanobiology LaboratoryNagoya University Graduate School of MedicineNagoyaJapan

Personalised recommendations