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ERK Signaling pp 203-221 | Cite as

Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors

  • Yannick Blum
  • Rafael D. Fritz
  • Hyunryul Ryu
  • Olivier PertzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1487)

Abstract

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.

Key words

Fluorescence resonance energy transfer (FRET) Ratiometric FRET imaging Biosensors EKAR2G ERK dynamics Cell-cell variability Signaling heterogeneity Cell fate Live-cell microscopy High throughput PC12 cells 

Notes

Acknowledgements

This work was supported by grants from the Human Frontier Science Program, the Swiss National Science Foundation, and the Novartis Foundation for medical-biological research.

References

  1. 1.
    O'Neill E, Kolch W (2004) Conferring specificity on the ubiquitous Raf/MEK signalling pathway. Br J Cancer 90:283–288CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wellbrock C, Karasarides M, Marais R (2004) The RAF proteins take centre stage. Nat Rev Mol Cell Biol 5:875–885CrossRefPubMedGoogle Scholar
  3. 3.
    Gehart H, Kumpf S, Ittner A et al (2010) MAPK signalling in cellular metabolism: stress or wellness? EMBO Rep 11:834–840CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Huang C, Jacobson K, Schaller MD (2004) MAP kinases and cell migration. J Cell Sci 117:4619–4628CrossRefPubMedGoogle Scholar
  5. 5.
    Downward J (2003) Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3:11–22CrossRefPubMedGoogle Scholar
  6. 6.
    O'Shaughnessy EC, Palani S, Collins JJ et al (2011) Tunable signal processing in synthetic MAP kinase cascades. Cell 144:119–131CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yoon S, Seger R (2006) The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24:21–44CrossRefPubMedGoogle Scholar
  8. 8.
    Albeck JG, Mills GB, Brugge JS (2013) Frequency-modulated pulses of ERK activity transmit quantitative proliferation signals. Mol Cell 49:249–261CrossRefPubMedGoogle Scholar
  9. 9.
    Aoki K, Kumagai Y, Sakurai A et al (2013) Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation. Mol Cell 52:529–540CrossRefPubMedGoogle Scholar
  10. 10.
    Fritz RD, Letzelter M, Reimann A et al (2013) A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Sci Signal 6(285):12CrossRefGoogle Scholar
  11. 11.
    Harvey CD, Ehrhardt AG, Cellurale C et al (2008) A genetically encoded fluorescent sensor of ERK activity. Proc Natl Acad Sci USA 105:19264–19269CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hirata E, Girotti MR, Viros A et al (2015) Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin beta1/FAK signaling. Cancer Cell 27:574–588CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Komatsu N, Aoki K, Yamada M et al (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell 22:4647–4656CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Mizuno R, Kamioka Y, Kabashima K et al (2014) In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines. J Exp Med 211:1123–1136CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ryu HCM, Dobrzynski M, Fey D et al (2015) Frequency modulation of ERK activation dynamics rewires cell fate. Mol Syst Biol 11:838CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hamers D, van Voorst VL, Borst JW et al (2014) Development of FRET biosensors for mammalian and plant systems. Protoplasma 251:333–347CrossRefPubMedGoogle Scholar
  17. 17.
    Dehmelt L, Bastiaens PI (2010) Spatial organization of intracellular communication: insights from imaging. Nat Rev Mol Cell Biol 11:440–452CrossRefPubMedGoogle Scholar
  18. 18.
    Heidecker G, Huleihel M, Cleveland JL et al (1990) Mutational activation of c-raf-1 and definition of the minimal transforming sequence. Mol Cell Biol 10:2503–2512CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Yan M, Templeton DJ (1994) Identification of 2 serine residues of MEK-1 that are differentially phosphorylated during activation by raf and MEK kinase. J Biol Chem 269:19067–19073PubMedGoogle Scholar
  20. 20.
    Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185CrossRefPubMedGoogle Scholar
  21. 21.
    Santos SD, Verveer PJ, Bastiaens PI (2007) Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate. Nat Cell Biol 9:324–330CrossRefPubMedGoogle Scholar
  22. 22.
    von Kriegsheim A, Baiocchi D, Birtwistle M et al (2009) Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 11:1458–1464CrossRefGoogle Scholar
  23. 23.
    Sasagawa S, Ozaki Y, Fujita K et al (2005) Prediction and validation of the distinct dynamics of transient and sustained ERK activation. Nat Cell Biol 7:365–373CrossRefPubMedGoogle Scholar
  24. 24.
    Davis HE, Morgan JR, Yarmush ML (2002) Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys Chem 97:159–172CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Yannick Blum
    • 1
    • 2
  • Rafael D. Fritz
    • 1
  • Hyunryul Ryu
    • 3
    • 4
    • 5
  • Olivier Pertz
    • 1
    • 2
    Email author
  1. 1.Department of BiomedicineUniversity of BaselBaselSwitzerland
  2. 2.Institute of Cell BiologyUniversity of BernBernSwitzerland
  3. 3.School of Mechanical and Aerospace EngineeringSeoul National UniversitySeoulRepublic of Korea
  4. 4.Institute of Advanced Machinery and DesignSeoul National UniversitySeoulRepublic of Korea
  5. 5.Research Laboratory of ElectronicsMassachusetts Institute of TechnologyCambridgeUSA

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