Optogenetic Control of Ras/Erk Signaling Using the Phy–PIF System

  • Alexander G. Goglia
  • Maxwell Z. Wilson
  • Daniel B. DiGiorno
  • Jared E. ToettcherEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1636)


The Ras/Erk signaling pathway plays a central role in diverse cellular processes ranging from development to immune cell activation to neural plasticity to cancer. In recent years, this pathway has been widely studied using live-cell fluorescent biosensors, revealing complex Erk dynamics that arise in many cellular contexts. Yet despite these high-resolution tools for measurement, the field has lacked analogous tools for control over Ras/Erk signaling in live cells. Here, we provide detailed methods for one such tool based on the optical control of Ras activity, which we call “Opto-SOS.” Expression of the Opto-SOS constructs can be coupled with a live-cell reporter of Erk activity to reveal highly quantitative input-to-output maps of the pathway. Detailed herein are protocols for expressing the Opto-SOS system in cultured cells, purifying the small molecule cofactor necessary for optical stimulation, imaging Erk responses using live-cell microscopy, and processing the imaging data to quantify Ras/Erk signaling dynamics.

Key words

Optogenetics Signal transduction Single-cell dynamics MAP kinase Ras Erk 



We thank Mohammad Seyedsayamdost for assistance and advice with HPLC purification. This work was supported by the NIH National Institute of Biomedical Imaging and Bioengineering (grant DP2EB024247 to J.E.T.), the NIH National Cancer Institute (fellowship F30CA206408 to A.G.G.), and a Princeton University Dean of Research Innovation Award to J.E.T.


  1. 1.
    Burack WR, Shaw AS (2005) Live cell imaging of ERK and MEK: simple binding equilibrium explains the regulated nucleocytoplasmic distribution of ERK. J Biol Chem 280:3832–3837CrossRefPubMedGoogle Scholar
  2. 2.
    Regot S, Hughey JJ, Bajar BT, Carrasco S, Covert MW (2014) High-sensitivity measurements of multiple kinase activities in live single cells. Cell 157:1724–1734CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Albeck JG, Mills GB, Brugge JS (2013) Frequency-modulated pulses of ERK activity transmit quantitative proliferation signals. Mol Cell 49:249–261CrossRefPubMedGoogle Scholar
  4. 4.
    Aoki K 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
  5. 5.
    Hiratsuka T et al (2015) Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin. Elife 4:e05178CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Mizuno R et al (2014) vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines. J Exp Med 211:1123–1136CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kumagai Y et al (2015) Heterogeneity in ERK activity as visualized by in vivo FRET imaging of mammary tumor cells developed in MMTV-Neu mice. Oncogene 34:1051–1057CrossRefPubMedGoogle Scholar
  8. 8.
    Huang CY, Ferrell JE Jr (1996) Ultrasensitivity in the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 93:10078–10083CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ferrell JE Jr, Machleder EM (1998) The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. Science 280:895–898CrossRefPubMedGoogle Scholar
  10. 10.
    Handly LN, Pilko A, Wollman R (2015) Paracrine communication maximizes cellular response fidelity in wound signaling. Elife 4:e09652CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Cohen-Saidon C, Cohen AA, Sigal A, Liron Y, Alon U (2009) Dynamics and variability of ERK2 response to EGF in individual living cells. Mol Cell 36:885–893CrossRefPubMedGoogle Scholar
  12. 12.
    Grusch M et al (2014) Spatio-temporally precise activation of engineered receptor tyrosine kinases by light. EMBO J 33:1713–1726CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhang K et al (2014) Light-mediated kinetic control reveals the temporal effect of the Raf/MEK/ERK pathway in PC12 cell neurite outgrowth. PLoS One 9:e92917CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Toettcher JE, Weiner OD, Lim WA (2013) Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155:1422–1434CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Toettcher JE, Gong D, Lim WA, Weiner OD (2011) Light-based feedback for controlling intracellular signaling dynamics. Nat Methods 8:837–839CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Toettcher JE, Gong D, Lim WA, Weiner OD (2011) Light control of plasma membrane recruitment using the Phy-PIF system. Methods Enzymol 497:409–423CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Levskaya A, Weiner OD, Lim WA, Voigt CA (2009) Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461:997–1001CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gautier A, Deiters A, Chin JW (2011) Light-activated kinases enable temporal dissection of signaling networks in living cells. J Am Chem Soc 133:2124–2127CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    O'Neill PR, Gautam N (2014) Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration. Mol Biol Cell 25:2305–2314CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Guntas G et al (2015) Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc Natl Acad Sci U S A 112:112–117CrossRefPubMedGoogle Scholar
  21. 21.
    Kennedy MJ et al (2010) Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods 7:973–975CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Tischer D, Weiner OD (2014) Illuminating cell signalling with optogenetic tools. Nat Rev Mol Cell Biol 15(8):551CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Buckley CE et al (2016) Reversible optogenetic control of subcellular protein localization in a live vertebrate embryo. Dev Cell 36:117–126CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kholodenko BN (2000) Negative feedback and ultrasensitivity can bring about oscillations in the mitogen-activated protein kinase cascades. Eur J Biochem 267:1583–1588CrossRefPubMedGoogle Scholar
  25. 25.
    Markevich NI, Hoek JB, Kholodenko BN (2004) Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades. J Cell Biol 164:353–359CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Boykevisch S et al (2006) Regulation of ras signaling dynamics by Sos-mediated positive feedback. Curr Biol 16:2173–2179CrossRefPubMedGoogle Scholar
  27. 27.
    Balan V et al (2006) Identification of novel in vivo Raf-1 phosphorylation sites mediating positive feedback Raf-1 regulation by extracellular signal-regulated kinase. Mol Biol Cell 17:1141–1153CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Fey D, Croucher DR, Kolch W, Kholodenko BN (2012) Crosstalk and signaling switches in mitogen-activated protein kinase cascades. Front Physiol 3:355CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Vogelstein B et al (2013) Cancer genome landscapes. Science 339:1546–1558CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Samatar AA, Poulikakos PI (2014) Targeting RAS-ERK signalling in cancer: promises and challenges. Nat Rev Drug Discov 13:928–942CrossRefPubMedGoogle Scholar
  31. 31.
    Rockwell NC, Su YS, Lagarias JC (2006) Phytochrome structure and signaling mechanisms. Annu Rev Plant Biol 57:837–858CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Khanna R et al (2004) A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. Plant Cell 16:3033–3044CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Gureasko J et al (2008) Membrane-dependent signal integration by the Ras activator Son of sevenless. Nat Struct Mol Biol 15:452–461CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Clarke S, Vogel JP, Deschenes RJ, Stock J (1988) Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci U S A 85:4643–4647CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Shankaran H et al (2009) Rapid and sustained nuclear-cytoplasmic ERK oscillations induced by epidermal growth factor. Mol Syst Biol 5:332CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Leung DW, Otomo C, Chory J, Rosen MK (2008) Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway. Proc Natl Acad Sci U S A 105:12797–12802CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Yang X, Jost AP, Weiner OD, Tang C (2013) A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast. Mol Biol Cell 24:2419–2430CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Davis HE, Rosinski M, Morgan JR, Yarmush ML (2004) Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation. Biophys J 86:1234–1242CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Mirzoeva OK et al (2009) Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition. Cancer Res 69:565–572CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Alexander G. Goglia
    • 1
    • 2
  • Maxwell Z. Wilson
    • 1
  • Daniel B. DiGiorno
    • 1
  • Jared E. Toettcher
    • 1
    Email author
  1. 1.Department of Molecular BiologyPrinceton UniversityPrincetonUSA
  2. 2.Robert Wood Johnson Medical SchoolRutgers UniversityNew BrunswickUSA

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