Rho GTPases pp 215-234 | Cite as

Multiplex Imaging of Rho Family GTPase Activities in Living Cells

  • Désirée Spiering
  • Louis HodgsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 827)


Here, we provide procedures for imaging the Rho GTPase biosensors in both single and multiplex acquisition modes. The multiplex approach enables the direct visualization of two biosensor readouts from a single living cell. Here, we take as an example a combination of the RhoA biosensor based on a CFP/YFP FRET modality and the Cdc42 biosensor based on organic dyes that change fluorescence as a function of the local solvent polarity. We list the required optical components as well as cellular manipulation techniques necessary to successfully image these two ratiometric biosensors in a single living cell.

Key words

Rho GTPase Dynamics Live-cell imaging FRET Biosensors Multiplex 



This work was funded by GM093121 (D.S. and L.H.) and “Sinsheimer Foundation Young Investigator Award” (L.H.).


  1. 1.
    Kraynov, V.S., Chamberlain, C., Bokoch, G.M., Schwartz, M.A., Slabaugh, S., and Hahn, K.M. (2000) Localized Rac activation dynamics visualized in living cells, Science 290, 333–337.PubMedCrossRefGoogle Scholar
  2. 2.
    Nalbant, P., Hodgson, L., Kraynov, V., Toutchkine, A., and Hahn, K.M. (2004) Activation of endogenous Cdc42 visualized in living cells, Science 305, 1615–1619.PubMedCrossRefGoogle Scholar
  3. 3.
    Pertz, O., Hodgson, L., Klemke, R.L., and Hahn, K.M. (2006) Spatiotemporal dynamics of RhoA activity in migrating cells, Nature 440, 1069–1072.PubMedCrossRefGoogle Scholar
  4. 4.
    Machacek, M., Hodgson, L., Welch, C., Elliott, H., Pertz, O., Nalbant, P., Abell, A., Johnson, G.L., Hahn, K.M., and Danuser, G. (2009) Coordination of Rho GTPase activities during cell protrusion, Nature 461, 99–103.PubMedCrossRefGoogle Scholar
  5. 5.
    Seth, A., Otomo, T., Yin, H.L., and Rosen, M.K. (2003) Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling, Biochemistry 42, 3997–4008.PubMedCrossRefGoogle Scholar
  6. 6.
    Ridley, A.J., and Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors, Cell 70, 389–399.PubMedCrossRefGoogle Scholar
  7. 7.
    Ridley, A.J., Paterson, H.F., Johnston, C.L., Diekmann, D., and Hall, A. (1992) The small GTP-binding protein rac regulates growth factor-induced membrane ruffling, Cell 70, 401–410.PubMedCrossRefGoogle Scholar
  8. 8.
    Zawistowski, J., Sabouri-Ghomi, M., Hodgson, L., Danuser, G., and Hahn, K. (2011) Differential activation of RhoA and RhoC in migrating cells, Nature (Submitted)Google Scholar
  9. 8.
    Garrett, S.C., Hodgson, L., Rybin, A., Toutchkine, A., Hahn, K.M., Lawrence, D.S., and Bresnick, A.R. (2008) A biosensor of S100A4 metastasis factor activation: inhibitor screening and cellular activation dynamics, Biochemistry 47, 986–996.PubMedCrossRefGoogle Scholar
  10. 9.
    Hodgson, L., Shen, F., and Hahn, K.M. (2010) Biosensors for characterizing the dynamics of rho family GTPases in living cells In: Bonifacino, J.S, Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds) Current Protocols in Cell Biology, Unit 14.11. Wiley, New York.Google Scholar
  11. 10.
    Gossen, M., and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters, Proc Natl Acad Sci USA 89, 5547–5551.PubMedCrossRefGoogle Scholar
  12. 11.
    Hodgson, L., Pertz, O., and Hahn, K. M. (2008) Design and optimization of genetically encoded fluorescent biosensors: GTPase biosensors, Methods Cell Biol 85, 63–81.PubMedCrossRefGoogle Scholar
  13. 12.
    Ouyang, M., Huang, H., Shaner, N. C., Remacle, A.G., Shiryaev, S.A., Strongin, A.Y., Tsien, R.Y., and Wang, Y. (2010) Simultaneous visualization of protumorigenic Src and MT1-MMP activities with fluorescence resonance energy transfer, Cancer Res 70, 2204–2212.PubMedCrossRefGoogle Scholar
  14. 13.
    Toutchkine, A., Nguyen, D.V., and Hahn, K.M. (2007) Merocyanine dyes with improved photostability, Org Lett 9, 2775–2777.PubMedCrossRefGoogle Scholar
  15. 14.
    Kaighn, M.E. (1973) Tissue Culture Methods and Applications. Academy Press, New York.Google Scholar
  16. 15.
    Robey, P.G., and Termine, J.D. (1985) Human bone cells in vitro. Calcif Tissue Int 37, 453–460.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics CenterAlbert Einstein College of Medicine of Yeshiva UniversityBronxUSA

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