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Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors

Nature Protocols volume 6, pages 1835–1846 (2011)Cite this article

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Abstract

Fluorescence resonance energy transfer (FRET)-based molecular biosensors serve as important tools for studying protein activity in live cells and have been widely used for this purpose over the past decade. However, FRET biosensors are rarely used in the context of the live organism because of the inherent high cellular complexity and imaging challenges associated with the three-dimensional environment. Here we provide a protocol for using single-chain intramolecular FRET-based biosensors in early development. We provide a general protocol for FRET ratio imaging in embryos, including the data-acquisition conditions and the algorithm for ratio image generation. We then use the pRaichu RacFRET biosensor to exemplify the adaptation and optimization of a particular biosensor for use in live zebrafish embryos. Once an optimized biosensor is available, the complete procedure, including introduction of the probes into embryos, imaging and data analysis, requires 2–3 d.

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Figure 1: The basic structure of a single-chain FRET-based biosensor.
Figure 2: Experimental flow.
Figure 3: Image acquisition.
Figure 4: Ratio image generation procedure.
Figure 5: Ratio imaging artifacts.
Figure 6: Basic controls used during FRET imaging setup.
Figure 7: Adaptation of the RacFRET biosensor for measurements in zebrafish embryos.
Figure 8: Controls used for measuring Rac activity.
Figure 9: Examples of FRET images in zebrafish.

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References

  1. Palmer, A.E., Qin, Y., Park, J.G. & McCombs, J.E. Design and application of genetically encoded biosensors. Trends Biotechnol. 29, 144–152 (2011).

    Article  CAS  Google Scholar 

  2. Merlot, S. & Firtel, R.A. Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways. J. Cell Sci. 116, 3471–3478 (2003).

    Article  CAS  Google Scholar 

  3. Devreotes, P. & Janetopoulos, C. Eukaryotic chemotaxis: distinctions between directional sensing and polarization. J. Biol. Chem. 278, 20445–20448 (2003).

    Article  CAS  Google Scholar 

  4. Tsien, R.Y. & Miyawaki, A. Seeing the machinery of live cells. Science (New York, NY) 280, 1954–1955 (1998).

    Article  CAS  Google Scholar 

  5. Jares-Erijman, E.A. & Jovin, T.M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).

    Article  CAS  Google Scholar 

  6. Kiyokawa, E., Hara, S., Nakamura, T. & Matsuda, M. Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells. Cancer Sci. 97, 8–15 (2006).

    Article  CAS  Google Scholar 

  7. VanEngelenburg, S.B. & Palmer, A.E. Fluorescent biosensors of protein function. Curr. Opin. Chem. Biol. 12, 60–65 (2008).

    Article  CAS  Google Scholar 

  8. Kraynov, V.S. et al. Localized Rac activation dynamics visualized in living cells. Science (New York, NY) 290, 333–337 (2000).

    Article  CAS  Google Scholar 

  9. Pertz, O., Hodgson, L., Klemke, R.L. & Hahn, K.M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006).

    Article  CAS  Google Scholar 

  10. Gaits, F. & Hahn, K. Shedding light on cell signaling: interpretation of FRET biosensors. Sci. STKE 2003, PE3 (2003).

    PubMed  Google Scholar 

  11. Aoki, K. & Matsuda, M. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat. Protoc. 4, 1623–1631 (2009).

    Article  CAS  Google Scholar 

  12. Esposito, A., Gralle, M., Dani, M.A., Lange, D. & Wouters, F.S. pHlameleons: a family of FRET-based protein sensors for quantitative pH imaging. Biochemistry 47, 13115–13126 (2008).

    Article  CAS  Google Scholar 

  13. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  CAS  Google Scholar 

  14. Palmer, A.E. & Tsien, R.Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protoc. 1, 1057–1065 (2006).

    Article  CAS  Google Scholar 

  15. Itoh, R.E. et al. 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, 6582–6591 (2002).

    Article  CAS  Google Scholar 

  16. Wang, Y. et al. Visualizing the mechanical activation of Src. Nature 434, 1040–1045 (2005).

    Article  CAS  Google Scholar 

  17. Fosbrink, M., Aye-Han, N.N., Cheong, R., Levchenko, A. & Zhang, J. Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor. Proc. Natl. Acad. Sci. USA 107, 5459–5464 (2010).

    Article  CAS  Google Scholar 

  18. Sato, M., Kawai, Y. & Umezawa, Y. Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells. Anal. Chem. 79, 2570–2575 (2007).

    Article  CAS  Google Scholar 

  19. Kiyokawa, E., Aoki, K., Nakamura, T. & Matsuda, M. Spatiotemporal regulation of small GTPases as revealed by probes based on the principle of Forster Resonance Energy Transfer (FRET): implications for signaling and pharmacology. Ann. Rev. Pharmacol. Toxicol. 51, 337–358 (2011).

    Article  CAS  Google Scholar 

  20. Kardash, E. et al. A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nat. Cell Biol. 12, 47–53; sup pp 1–11 (2010).

    Article  CAS  Google Scholar 

  21. Miyagi, C. et al. STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension. J. Cell Biol. 166, 975–981 (2004).

    Article  CAS  Google Scholar 

  22. Stockholm, D. et al. Imaging calpain protease activity by multiphoton FRET in living mice. J. Mol. Biol. 346, 215–222 (2005).

    Article  CAS  Google Scholar 

  23. Blaser, H. et al. Migration of zebrafish primordial germ cells: a role for Myosin contraction and cytoplasmic flow. Dev. Cell 11, 613–627 (2006).

    Article  CAS  Google Scholar 

  24. Kamiyama, D. & Chiba, A. Endogenous activation patterns of Cdc42 GTPase within Drosophila embryos. Science (New York, NY) 324, 1338–1340 (2009).

    Article  CAS  Google Scholar 

  25. Wang, X., He, L., Wu, Y.I., Hahn, K.M. & Montell, D.J. Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat. Cell Biol. 12, 591–597 (2010).

    Article  CAS  Google Scholar 

  26. Horikawa, K. et al. Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano. Nat. Methods 7, 729–732 (2010).

    Article  CAS  Google Scholar 

  27. Xia, Z. & Liu, Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395–2402 (2001).

    Article  CAS  Google Scholar 

  28. Nakamura, T., Aoki, K. & Matsuda, M. Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes. Methods 37, 146–153 (2005).

    Article  CAS  Google Scholar 

  29. Köprunner, M., Thisse, C., Thisse, B. & Raz, E. A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev. 15, 2877–2885 (2001).

    PubMed  PubMed Central  Google Scholar 

  30. Goedhart, J. & Kremers, G.-J. in Laboratory Techniques in Biochemistry and Molecular Biology, FRET and FLIM Techniques, Vol. 33 (ed. Gadella, T.W.) Ch. 5, 171–223 (Elsevier, 2009).

  31. Nguyen, A.W. & Daugherty, P.S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).

    Article  CAS  Google Scholar 

  32. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  Google Scholar 

  33. Ouyang, M., Sun, J., Chien, S. & Wang, Y. Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors. Proc. Natl. Acad. Sci. USA 105, 14353–14358 (2008).

    Article  CAS  Google Scholar 

  34. Diekmann, D. et al. Bcr encodes a GTPase-activating protein for p21rac. Nature 351, 400–402 (1991).

    Article  CAS  Google Scholar 

  35. Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of zebrafish (Danio rerio), 4th edn. (University of Oregon Press, 2000).

  36. Rosen, J.N., Sweeney, M.F. & Mably, J.D. Microinjection of zebrafish embryos to analyze gene function. J. Vis. Exp. published online, doi:10.3791/1115 (9 March 2009).

  37. Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the Max Planck Society. We thank M. Reichman-Fried for critical reading of the manuscript and E.-M. Messerschmidt for technical assistance. We also thank M. Matsuda and P. Daugherty for sharing the constructs.

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Authors and Affiliations

  1. Institute for Cell Biology, Center for Molecular Biology of Inflammation, University of Münster, Münster, Germany

    Elena Kardash, Jan Bandemer & Erez Raz

Authors
  1. Elena Kardash
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  2. Jan Bandemer
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  3. Erez Raz
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Contributions

E.K. designed and performed the experiments, analyzed the data and wrote the paper. J.B. designed and performed the experiments, analyzed the data and wrote the paper. E.R. supervised the project, designed the experiments and wrote the paper.

Corresponding author

Correspondence to Erez Raz.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Raw data captured in the YFP channel, of a cell expressing SECFP-YPet construct with the histogram showing the pixel intensity distribution. The maximum intensity value detected by the camera is 4095. To generate the histogram, the 16 bit image is first converted to 32 bit using the command: Image > Image Type > 32 bit from the ImageJ menu. To obtain the histogram, the command: Analyse > Histogram was used and the desired range was defined in the Histogram dialog box. (JPG 460 kb)

Supplementary Data 1

rawCFP image. The CFP emission channel of the somatic cell expressing RacFRET-noCT construct. (It is recommended that this file is viewed using ImageJ, a public domain Java image processing program available at http://rsbweb.nih.gov/ij/) (TIFF 51 kb)

Supplementary Data 2

rawYFP image. The YFP emission channel of the somatic cell expressing RacFRET-noCT construct. (It is recommended that this file is viewed using ImageJ, a public domain Java image processing program available at http://rsbweb.nih.gov/ij/) (TIFF 51 kb)

Supplementary Table 1

List of example DNA constructs used in this protocol (DOC 964 kb)

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Kardash, E., Bandemer, J. & Raz, E. Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nat Protoc 6, 1835–1846 (2011). https://doi.org/10.1038/nprot.2011.395

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