Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors

  • Charlene Depry
  • Sohum Mehta
  • Jin Zhang
Invited Review


Cells rely on a complex, interconnected network of signaling pathways to sense and interpret changes in their extracellular environment. The development of genetically encoded fluorescent protein (FP)-based biosensors has made it possible for researchers to directly observe and characterize the spatiotemporal dynamics of these intracellular signaling pathways in living cells. However, detailed information regarding the precise temporal and spatial relationships between intersecting pathways is often lost when individual signaling events are monitored in isolation. As the development of biosensor technology continues to advance, it is becoming increasingly feasible to image multiple FP-based biosensors concurrently, permitting greater insights into the intricate coordination of intracellular signaling networks by enabling parallel monitoring of distinct signaling events within the same cell. In this review, we discuss several strategies for multiplexed imaging of FP-based biosensors, while also underscoring some of the challenges associated with these techniques and highlighting additional avenues that could lead to further improvements in parallel monitoring of intracellular signaling events.


Co-imaging FRET Signal transduction Microscopy Biosensor 



We wish to thank members of the Zhang lab for helpful discussions, in particular Fabian Hertel for comments on the manuscript. This work is supported by the National Institutes of Health (R01DK073368 and DP1CA174423 to J.Z.).

Conflict of interest

The authors declare they have no competing financial interests.


  1. 1.
    Ai HW, Hazelwood KL, Davidson MW, Campbell RE (2008) Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat Methods 5:401–403PubMedCrossRefGoogle Scholar
  2. 2.
    Akerboom J, Rivera JD, Guilbe MM, Malave EC, Hernandez HH, Tian L, Hires SA, Marvin JS, Looger LL, Schreiter ER (2009) Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J Biol Chem 284:6455–6464PubMedCrossRefGoogle Scholar
  3. 3.
    Ananthanarayanan B, Fosbrink M, Rahdar M, Zhang J (2007) Live-cell molecular analysis of Akt activation reveals roles for activation loop phosphorylation. J Biol Chem 282:36634–36641PubMedCrossRefGoogle Scholar
  4. 4.
    Ananthanarayanan B, Ni Q, Zhang J (2005) Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity. Proc Natl Acad Sci U S A 102:15081–15086PubMedCrossRefGoogle Scholar
  5. 5.
    Aye-Han NN, Allen MD, Ni Q, Zhang J (2012) Parallel tracking of cAMP and PKA signaling dynamics in living cells with FRET-based fluorescent biosensors. Mol Biosyst 8:1435–1440PubMedCrossRefGoogle Scholar
  6. 6.
    Chiu VK, Bivona T, Hach A, Sajous JB, Silletti J, Wiener H, 2nd Johnson RL, Cox AD, Philips MR (2002) Ras signalling on the endoplasmic reticulum and the Golgi. Nat Cell Biol 4:343–350PubMedGoogle Scholar
  7. 7.
    Choi Y, Kim K, Hong S, Kim H, Kwon YJ, Song R (2011) Intracellular protein target detection by quantum dots optimized for live cell imaging. Bioconjug Chem 22:1576–1586PubMedCrossRefGoogle Scholar
  8. 8.
    Cicchetti G, Biernacki M, Farquharson J, Allen PG (2004) A ratiometric expressible FRET sensor for phosphoinositides displays a signal change in highly dynamic membrane structures in fibroblasts. Biochemistry 43:1939–1949PubMedCrossRefGoogle Scholar
  9. 9.
    Cohen P (2002) Protein kinases—the major drug targets of the twenty-first century. Nat Rev Drug Discov 1:309–315PubMedCrossRefGoogle Scholar
  10. 10.
    DeMarinis RM, Katerinopoulos HE, Muirhead KA (1990) New tetracarboxylate compounds as fluorescent intracellular calcium indicators. Biochem Meth 112:381Google Scholar
  11. 11.
    Depry C, Allen MD, Zhang J (2011) Visualization of PKA activity in plasma membrane microdomains. Mol Biosyst 7:52–58PubMedCrossRefGoogle Scholar
  12. 12.
    Depry C, Zhang J (2010) Visualization of kinase activity with FRET-based activity biosensors. Curr Protoc Mol Biol Chapter 18:Unit 18.15Google Scholar
  13. 13.
    Deuschle K, Okumoto S, Fehr M, Looger LL, Kozhukh L, Frommer WB (2005) Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering. Protein Sci 14:2304–2314PubMedCrossRefGoogle Scholar
  14. 14.
    Ding Y, Ai HW, Hoi H, Campbell RE (2011) Forster resonance energy transfer-based biosensors for multiparameter ratiometric imaging of Ca2+ dynamics and caspase-3 activity in single cells. Anal Chem 83:9687–9693PubMedCrossRefGoogle Scholar
  15. 15.
    DiPilato LM, Cheng X, Zhang J (2004) Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci U S A 101:16513–16518PubMedCrossRefGoogle Scholar
  16. 16.
    DiPilato LM, Zhang J (2009) The role of membrane microdomains in shaping beta2-adrenergic receptor-mediated cAMP dynamics. Mol Biosyst 5:832–837PubMedCrossRefGoogle Scholar
  17. 17.
    Fehr M, Lalonde S, Lager I, Wolff MW, Frommer WB (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J Biol Chem 278:19127–19133PubMedCrossRefGoogle Scholar
  18. 18.
    Frommer WB, Davidson MW, Campbell RE (2009) Genetically encoded biosensors based on engineered fluorescent proteins. Chem Soc Rev 38:2833–2841PubMedCrossRefGoogle Scholar
  19. 19.
    Gallegos LL, Kunkel MT, Newton AC (2006) Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling. J Biol Chem 281:30947–30956PubMedCrossRefGoogle Scholar
  20. 20.
    Garini Y, Young IT, McNamara G (2006) Spectral imaging: principles and applications. Cytometry A 69:735–747PubMedGoogle Scholar
  21. 21.
    Gerbino A, Ruder WC, Curci S, Pozzan T, Zaccolo M, Hofer AM (2005) Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations. J Cell Biol 171:303–312PubMedCrossRefGoogle Scholar
  22. 22.
    Grant SK (2009) Therapeutic protein kinase inhibitors. Cell Mol Life Sci 66:1163–1177PubMedCrossRefGoogle Scholar
  23. 23.
    Halet G (2005) Imaging phosphoinositide dynamics using GFP-tagged protein domains. Biol Cell 97:501–518PubMedCrossRefGoogle Scholar
  24. 24.
    Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, Svoboda K (2008) A genetically encoded fluorescent sensor of ERK activity. Proc Natl Acad Sci U S A 105:19264–19269PubMedCrossRefGoogle Scholar
  25. 25.
    Hiraoka Y, Shimi T, Haraguchi T (2002) Multispectral imaging fluorescence microscopy for living cells. Cell Struct Funct 27:367–374PubMedCrossRefGoogle Scholar
  26. 26.
    Honda A, Sawyer CL, Cawley SM, Dostmann WR (2005) Cygnets: in vivo characterization of novel cGMP indicators and in vivo imaging of intracellular cGMP. Methods Mol Biol 307:27–43PubMedGoogle Scholar
  27. 27.
    Imamura H, Nhat KP, Togawa H, Saito K, Iino R, Kato-Yamada Y, Nagai T, Noji H (2009) Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc Natl Acad Sci U S A 106:15651–15656PubMedCrossRefGoogle Scholar
  28. 28.
    Itoh RE, Kurokawa K, Fujioka A, Sharma A, Mayer BJ, Matsuda M (2005) A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes. Exp Cell Res 307:142–152PubMedCrossRefGoogle Scholar
  29. 29.
    Itoh RE, Kurokawa K, Ohba Y, Yoshizaki H, Mochizuki N, Matsuda M (2002) Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells. Mol Cell Biol 22:6582–6591PubMedCrossRefGoogle Scholar
  30. 30.
    Johnson LN (2009) Protein kinase inhibitors: contributions from structure to clinical compounds. Q Rev Biophys 42:1–40PubMedCrossRefGoogle Scholar
  31. 31.
    Kawai H, Suzuki T, Kobayashi T, Sakurai H, Ohata H, Honda K, Momose K, Namekata I, Tanaka H, Shigenobu K, Nakamura R, Hayakawa T, Kawanishi T (2005) Simultaneous real-time detection of initiator- and effector-caspase activation by double fluorescence resonance energy transfer analysis. J Pharmacol Sci 97:361–368PubMedCrossRefGoogle Scholar
  32. 32.
    Komatsu N, Aoki K, Yamada M, Yukinaga H, Fujita Y, Kamioka Y, Matsuda M (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell 22:4647–4656PubMedCrossRefGoogle Scholar
  33. 33.
    Kunkel MT, Ni Q, Tsien RY, Zhang J, Newton AC (2005) Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter. J Biol Chem 280:5581–5587PubMedCrossRefGoogle Scholar
  34. 34.
    Kunkel MT, Toker A, Tsien RY, Newton AC (2007) Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter. J Biol Chem 282:6733–6742PubMedCrossRefGoogle Scholar
  35. 35.
    Kurebayashi N, Harkins AB, Baylor SM (1993) Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers. Biophys J 64:1934–1960PubMedCrossRefGoogle Scholar
  36. 36.
    Landa LR Jr, Harbeck M, Kaihara K, Chepurny O, Kitiphongspattana K, Graf O, Nikolaev VO, Lohse MJ, Holz GG, Roe MW (2005) Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 beta-cell line. J Biol Chem 280:31294–31302PubMedCrossRefGoogle Scholar
  37. 37.
    Li Y, Tsien RW (2012) pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity. Nat Neurosci 15:1047–1053PubMedCrossRefGoogle Scholar
  38. 38.
    Machacek M, Hodgson L, Welch C, Elliott H, Pertz O, Nalbant P, Abell A, Johnson GL, Hahn KM, Danuser G (2009) Coordination of Rho GTPase activities during cell protrusion. Nature 461:99–103PubMedCrossRefGoogle Scholar
  39. 39.
    Mank M, Griesbeck O (2008) Genetically encoded calcium indicators. Chem Rev 108:1550–1564PubMedCrossRefGoogle Scholar
  40. 40.
    Mehta S, Zhang J (2011) Reporting from the field: genetically encoded fluorescent reporters uncover signaling dynamics in living biological systems. Annu Rev Biochem 80:375–401PubMedCrossRefGoogle Scholar
  41. 41.
    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887PubMedCrossRefGoogle Scholar
  42. 42.
    Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A, Matsuda M (2001) Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411:1065–1068PubMedCrossRefGoogle Scholar
  43. 43.
    Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A 98:3197–3202PubMedCrossRefGoogle Scholar
  44. 44.
    Nausch LW, Ledoux J, Bonev AD, Nelson MT, Dostmann WR (2008) Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors. Proc Natl Acad Sci U S A 105:365–370PubMedCrossRefGoogle Scholar
  45. 45.
    Newman RH, Fosbrink MD, Zhang J (2011) Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111:3614–3666PubMedCrossRefGoogle Scholar
  46. 46.
    Newman RH, Zhang J (2008) Visualization of phosphatase activity in living cells with a FRET-based calcineurin activity sensor. Mol Biosyst 4:496–501PubMedCrossRefGoogle Scholar
  47. 47.
    Ni Q, Ganesan A, Aye-Han NN, Gao X, Allen MD, Levchenko A, Zhang J (2011) Signaling diversity of PKA achieved via a Ca2+-cAMP-PKA oscillatory circuit. Nat Chem Biol 7:34–40PubMedCrossRefGoogle Scholar
  48. 48.
    Niino Y, Hotta K, Oka K (2009) Simultaneous live cell imaging using dual FRET sensors with a single excitation light. PLoS One 4:e6036PubMedCrossRefGoogle Scholar
  49. 49.
    Niino Y, Hotta K, Oka K (2010) Blue fluorescent cGMP sensor for multiparameter fluorescence imaging. PLoS One 5:e9164PubMedCrossRefGoogle Scholar
  50. 50.
    Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218PubMedCrossRefGoogle Scholar
  51. 51.
    Nikolaev VO, Bunemann M, Schmitteckert E, Lohse MJ, Engelhardt S (2006) Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ Res 99:1084–1091PubMedCrossRefGoogle Scholar
  52. 52.
    Nikolaev VO, Gambaryan S, Lohse MJ (2006) Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods 3:23–25PubMedCrossRefGoogle Scholar
  53. 53.
    Nishioka T, Aoki K, Hikake K, Yoshizaki H, Kiyokawa E, Matsuda M (2008) Rapid turnover rate of phosphoinositides at the front of migrating MDCK cells. Mol Biol Cell 19:4213–4223PubMedCrossRefGoogle Scholar
  54. 54.
    Ouyang M, Huang H, Shaner NC, Remacle AG, Shiryaev SA, Strongin AY, Tsien RY, Wang Y (2010) Simultaneous visualization of protumorigenic Src and MT1-MMP activities with fluorescence resonance energy transfer. Cancer Res 70:2204–2212PubMedCrossRefGoogle Scholar
  55. 55.
    Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt M, Kanai AJ, McLaughlin MK, Pitt BR, Levitan ES (2000) Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Natl Acad Sci U S A 97:477–482PubMedCrossRefGoogle Scholar
  56. 56.
    Piljic A, Schultz C (2008) Simultaneous recording of multiple cellular events by FRET. ACS Chem Biol 3:156–160PubMedCrossRefGoogle Scholar
  57. 57.
    Russwurm M, Mullershausen F, Friebe A, Jager R, Russwurm C, Koesling D (2007) Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach. Biochem J 407:69–77PubMedCrossRefGoogle Scholar
  58. 58.
    Sample V, DiPilato LM, Yang JH, Ni Q, Saucerman JJ, Zhang J (2012) Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP. Nat Chem Biol 8:375–382PubMedCrossRefGoogle Scholar
  59. 59.
    Sato M, Hida N, Ozawa T, Umezawa Y (2000) Fluorescent indicators for cyclic GMP based on cyclic GMP-dependent protein kinase Ialpha and green fluorescent proteins. Anal Chem 72:5918–5924PubMedCrossRefGoogle Scholar
  60. 60.
    Sato M, Nakajima T, Goto M, Umezawa Y (2006) Cell-based indicator to visualize picomolar dynamics of nitric oxide release from living cells. Anal Chem 78:8175–8182PubMedCrossRefGoogle Scholar
  61. 61.
    Shelly M, Lim BK, Cancedda L, Heilshorn SC, Gao H, Poo MM (2010) Local and long-range reciprocal regulation of cAMP and cGMP in axon/dendrite formation. Science 327:547–552PubMedCrossRefGoogle Scholar
  62. 62.
    Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6:875–881PubMedCrossRefGoogle Scholar
  63. 63.
    Tsou P, Zheng B, Hsu CH, Sasaki AT, Cantley LC (2011) A fluorescent reporter of AMPK activity and cellular energy stress. Cell Metab 13:476–486PubMedCrossRefGoogle Scholar
  64. 64.
    van der Wal J, Habets R, Varnai P, Balla T, Jalink K (2001) Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer. J Biol Chem 276:15337–15344PubMedCrossRefGoogle Scholar
  65. 65.
    Varnai P, Balla T (2006) Live cell imaging of phosphoinositide dynamics with fluorescent protein domains. Biochim Biophys Acta 1761:957–967PubMedCrossRefGoogle Scholar
  66. 66.
    Violin JD, Zhang J, Tsien RY, Newton AC (2003) A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 161:899–909PubMedCrossRefGoogle Scholar
  67. 67.
    Welch CM, Elliott H, Danuser G, Hahn KM (2011) Imaging the coordination of multiple signalling activities in living cells. Nat Rev Mol Cell Biol 12:749–756PubMedCrossRefGoogle Scholar
  68. 68.
    Wong K, Pertz O, Hahn K, Bourne H (2006) Neutrophil polarization: spatiotemporal dynamics of RhoA activity support a self-organizing mechanism. Proc Natl Acad Sci U S A 103:3639–3644PubMedCrossRefGoogle Scholar
  69. 69.
    Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, Mochizuki N, Nagashima K, Matsuda M (2003) Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J Cell Biol 162:223–232PubMedCrossRefGoogle Scholar
  70. 70.
    Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2:25–29PubMedCrossRefGoogle Scholar
  71. 71.
    Zimmermann T, Rietdorf J, Pepperkok R (2003) Spectral imaging and its applications in live cell microscopy. FEBS Lett 546:87–92PubMedCrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  1. 1.Department of Pharmacology and Molecular SciencesThe Johns Hopkins University School of MedicineBaltimoreUSA
  2. 2.The Solomon H. Snyder Department of Neuroscience and Department of OncologyThe Johns Hopkins University School of MedicineBaltimoreUSA

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