Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
Pertz, O., Hodgson, L., Klemke, R.L., and Hahn, K.M. (2006) Spatiotemporal dynamics of RhoA activity in migrating cells, Nature 440, 1069–1072.
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.
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.
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.
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.
Zawistowski, J., Sabouri-Ghomi, M., Hodgson, L., Danuser, G., and Hahn, K. (2011) Differential activation of RhoA and RhoC in migrating cells, Nature (Submitted)
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.
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.
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.
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.
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.
Toutchkine, A., Nguyen, D.V., and Hahn, K.M. (2007) Merocyanine dyes with improved photostability, Org Lett 9, 2775–2777.
Kaighn, M.E. (1973) Tissue Culture Methods and Applications. Academy Press, New York.
Robey, P.G., and Termine, J.D. (1985) Human bone cells in vitro. Calcif Tissue Int 37, 453–460.
Acknowledgments
This work was funded by GM093121 (D.S. and L.H.) and “Sinsheimer Foundation Young Investigator Award” (L.H.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Spiering, D., Hodgson, L. (2012). Multiplex Imaging of Rho Family GTPase Activities in Living Cells. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 827. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-442-1_15
Download citation
DOI: https://doi.org/10.1007/978-1-61779-442-1_15
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-441-4
Online ISBN: 978-1-61779-442-1
eBook Packages: Springer Protocols