Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Multiplexed GTPase and GEF biosensor imaging enables network connectivity analysis


Here we generate fluorescence resonance energy transfer biosensors for guanine exchange factors (GEFs) by inserting a fluorescent protein pair in a structural ‘hinge’ common to many GEFs. Fluorescent biosensors can map the activation of signaling molecules in space and time, but it has not been possible to quantify how different activation events affect one another or contribute to a specific cell behavior. By imaging the GEF biosensors in the same cells as red-shifted biosensors of Rho GTPases, we can apply partial correlation analysis to parse out the extent to which each GEF contributes to the activation of a specific GTPase in regulating cell movement. Through analysis of spontaneous cell protrusion events, we identify when and where the GEF Asef regulates the GTPases Cdc42 and Rac1 to control cell edge dynamics. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Biosensors for Asef and Vav2.
Fig. 2: Biosensors for RhoGEFs without high-resolution structures.
Fig. 3: RhoGEF and GTPase activity in living cells.
Fig. 4: Correlation of protein activities with edge motion.
Fig. 5: Multiplex imaging of two protein activities.
Fig. 6: Partial correlation analysis.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Figures 46 have associated raw data used to produce the correlation plots.

Code availability

All code used for the data analysis was written in MATLAB 2014b. All the code can be downloaded from the Danuser Lab github: under Biosensor, Windowing-Protrusion and Time-Series-Tools repositories.


  1. 1.

    Devreotes, P. & Horwitz, A. R. Signaling networks that regulate cell migration. Cold Spring Harb. Perspect. Biol. 7, a005959 (2015).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Machacek, M. et al. Coordination of Rho GTPase activities during cell protrusion. Nature 461, 99–103 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Yang, H. W., Collins, S. R. & Meyer, T. Locally excitable Cdc42 signals steer cells during chemotaxis. Nat. Cell Biol. 18, 191–201 (2016).

    CAS  PubMed  Google Scholar 

  5. 5.

    Zawistowski, J. S., Sabouri-Ghomi, M., Danuser, G., Hahn, K. M. & Hodgson, L. A RhoC biosensor reveals differences in the activation kinetics of RhoA and RhoC in migrating cells. PLoS ONE 8, e79877 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Hodgson, L. et al. FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions. Nat. Chem. Biol. 12, 802–809 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Rossman, K. L., Der, C. J. & Sondek, J. GEF means go: turning on Rho GTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6, 167–180 (2005).

    CAS  PubMed  Google Scholar 

  8. 8.

    Hall, A. Rho family GTPases. Biochem. Soc. Trans. 40, 1378–1382 (2012).

    CAS  PubMed  Google Scholar 

  9. 9.

    Mitin, N. et al. Release of autoinhibition of ASEF by APC leads to Cdc42 activation and tumor suppression. Nat. Struct. Mol. Biol. 14, 814–823 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Slattery, S. D. & Hahn, K. M. A high-content assay for biosensor validation and for examining stimuli that affect biosensor activity. Curr. Protoc. Cell Biol. 65, 11–31 (2014).

    Google Scholar 

  11. 11.

    Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    CAS  PubMed  Google Scholar 

  12. 12.

    Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    CAS  PubMed  Google Scholar 

  13. 13.

    Markwardt, M. L. et al. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLoS ONE 6, e17896 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Xia, N. S. et al. Bioluminescence of Aequorea macrodactyla, a common jellyfish species in the East China Sea. Mar. Biotechnol. 4, 155–162 (2002).

    CAS  PubMed  Google Scholar 

  15. 15.

    Ai, H. W., Henderson, J. N., Remington, S. J. & Campbell, R. E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem. J. 400, 531–540 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

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

    CAS  PubMed  Google Scholar 

  17. 17.

    Itoh, R. E. et al. Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF. J. Cell Sci. 121, 2635–2642 (2008).

    CAS  PubMed  Google Scholar 

  18. 18.

    Yu, B. et al. Structural and energetic mechanisms of cooperative autoinhibition and activation of Vav1. Cell 140, 246–256 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Aghazadeh, B., Lowry, W. E., Huang, X. Y. & Rosen, M. K. Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 102, 625–633 (2000).

    CAS  PubMed  Google Scholar 

  20. 20.

    Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S. & Bustelo, X. R. Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385, 169–172 (1997).

    CAS  PubMed  Google Scholar 

  21. 21.

    Barreira, M. et al. The C-terminal SH3 domain contributes to the intramolecular inhibition of Vav family proteins. Sci. Signal 7, ra35 (2014).

    PubMed  Google Scholar 

  22. 22.

    Yohe, M. E. et al. Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif. J. Biol. Chem. 282, 13813–13823 (2007).

    CAS  PubMed  Google Scholar 

  23. 23.

    Yohe, M. E., Rossman, K. & Sondek, J. Role of the C-terminal SH3 domain and N-terminal tyrosine phosphorylation in regulation of Tim and related Dbl-family proteins. Biochemistry 47, 6827–6839 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Xu, Z., Gakhar, L., Bain, F. E., Spies, M. & Fuentes, E. J. The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain. J. Biol. Chem. 292, 17777–17793 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Chen, Z., Guo, L., Sprang, S. R. & Sternweis, P. C. Modulation of a GEF switch: autoinhibition of the intrinsic guanine nucleotide exchange activity of p115-RhoGEF. Protein Sci. 20, 107–117 (2011).

    CAS  PubMed  Google Scholar 

  26. 26.

    Lambert, J. M. et al. Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism. Nat. Cell Biol. 4, 621–625 (2002).

    CAS  PubMed  Google Scholar 

  27. 27.

    Miyamoto, Y., Yamauchi, J., Tanoue, A., Wu, C. & Mobley, W. C. TrkB binds and tyrosine-phosphorylates Tiam1, leading to activation of Rac1 and induction of changes in cellular morphology. Proc. Natl Acad. Sci. USA 103, 10444–10449 (2006).

    CAS  PubMed  Google Scholar 

  28. 28.

    Servitja, J. M., Marinissen, M. J., Sodhi, A., Bustelo, X. R. & Gutkind, J. S. Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src. J. Biol. Chem. 278, 34339–34346 (2003).

    CAS  PubMed  Google Scholar 

  29. 29.

    Tolias, K. F. et al. The Rac1 guanine nucleotide exchange factor Tiam1 mediates EphB receptor-dependent dendritic spine development. Proc. Natl Acad. Sci. USA 104, 7265–7270 (2007).

    CAS  PubMed  Google Scholar 

  30. 30.

    Suzuki, N., Nakamura, S., Mano, H. & Kozasa, T. Galpha 12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF. Proc. Natl Acad. Sci. USA 100, 733–738 (2003).

    CAS  PubMed  Google Scholar 

  31. 31.

    Feng, Q. et al. Cool-1 functions as an essential regulatory node for EGF receptor- and Src-mediated cell growth. Nat. Cell Biol. 8, 945–956 (2006).

    CAS  PubMed  Google Scholar 

  32. 32.

    Jaiswal, M. et al. Mechanistic insights into specificity, activity, and regulatory elements of the regulator of G-protein signaling (RGS)-containing Rho-specific guanine nucleotide exchange factors (GEFs) p115, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). J. Biol. Chem. 286, 18202–18212 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Nalbant, P., Hodgson, L., Kraynov, V., Toutchkine, A. & Hahn, K. M. Activation of endogenous Cdc42 visualized in living cells. Science 305, 1615–1619 (2004).

    CAS  PubMed  Google Scholar 

  34. 34.

    Herrington, K. A. et al. Spatial analysis of Cdc42 activity reveals a role for plasma membrane-associated Cdc42 in centrosome regulation. Mol. Biol. Cell 28, 2135–2145 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Mendoza, M. C., Vilela, M., Juarez, J. E., Blenis, J. & Danuser, G. ERK reinforces actin polymerization to power persistent edge protrusion during motility. Sci. Signal 8, ra47 (2015).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Lee, K. et al. Functional hierarchy of redundant actin assembly factors revealed by fine-grained registration of intrinsic image fluctuations. Cell Syst. 1, 37–50 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Ji, L., Lim, J. & Danuser, G. Fluctuations of intracellular forces during cell protrusion. Nat. Cell Biol. 10, 1393–1400 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Shcherbakova, D. M., Hink, M. A., Joosen, L., Gadella, T. W. & Verkhusha, V. V. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J. Am. Chem. Soc. 134, 7913–7923 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    CAS  PubMed  Google Scholar 

  40. 40.

    Nishimura, T. et al. PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1. Nat. Cell Biol. 7, 270–277 (2005).

    CAS  PubMed  Google Scholar 

  41. 41.

    Martin, K. et al. Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics. Sci. Rep. 6, 21901 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Kuo, J. C., Han, X., Hsiao, C. T., Yates, J. R. 3rd & Waterman, C. M. Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for beta-Pix in negative regulation of focal adhesion maturation. Nat. Cell Biol. 13, 383–393 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Goicoechea, S. M., Awadia, S. & Garcia-Mata, R. I’m coming to GEF you: regulation of RhoGEFs during cell migration. Cell Adh. Migr. 8, 535–549 (2014).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Lawson, C. D. & Ridley, A. J. Rho GTPase signaling complexes in cell migration and invasion. J. Cell Biol. 217, 447–457 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Azoitei, M. L. et al. Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways. J. Cell Biol. 218, 3077–3097 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Whitlow, M. et al. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng. 6, 989–995 (1993).

    CAS  PubMed  Google Scholar 

  47. 47.

    Kuhlman, B., Yang, H. Y., Boice, J. A., Fairman, R. & Raleigh, D. P. An exceptionally stable helix from the ribosomal protein L9: implications for protein folding and stability. J. Mol. Biol. 270, 640–647 (1997).

    CAS  PubMed  Google Scholar 

  48. 48.

    Kim, J.H. et al. High cleavage efficiency of a 2A peptide derived from porcine Teschovirus-1 in human cell lines, zebrafish and MICE. PLoS ONE 6, e18556 (2011).

  49. 49.

    Lindenburg, L. H., Hessels, A. M., Ebberink, E. H., Arts, R. & Merkx, M. Robust red FRET sensors using self-associating fluorescent domains. ACS Chem. Biol. 8, 2133–2139 (2013).

    CAS  PubMed  Google Scholar 

  50. 50.

    Fellmann, C. et al. An optimized microRNA backbone for effective single-copy RNAi. Cell Rep. 5, 1704–1713 (2013).

    CAS  PubMed  Google Scholar 

  51. 51.

    Shcherbakova, D. M. & Verkhusha, V. V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods 10, 751–754 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Vilela, M. et al. Fluctuation analysis of activity biosensor images for the study of information flow in signaling pathways. Methods Enzymol. 519, 253–276 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Huang, N. E. et al. The empirical mode decomposition and the Hilbert spectrum for nonlinear and non-stationary time series analysis. Proc. R. Soc. A. 454, 903–995 (1998).

    Google Scholar 

  54. 54.

    Zoubir, A. M. & Iskander, D. R. Bootstrap Techniques for Signal Processing (Cambridge Univ. Press, 2004).

  55. 55.

    Bailey, N. T. J. Statistical Methods in Biology 3rd edn (Cambridge Univ. Press, 1995).

Download references


Grant nos. R01-GM062299 (J.S.), R01 GM071868 (G.D.) and R35GM122596 (K.M.H.) supported this work. D.J.M. was supported by funding from the Leukemia and Lymphoma Society. J.H. was supported by a Human Frontiers in Sciences Program. G.G. was supported by the ‘Integrated Training in Cancer Model Systems’ Training Program, T32CA009156. We thank the Calabrese laboratory and F. Pimenta for the shRNA expression vector.

Author information




D.J.M., J.S. and K.M.H. designed the biosensors. D.J.M. carried out all experiments, except for work by J.R. in building Tiam1, Tim, B-Pix and LARG biosensors. G.G. and M.A. contributed to initial design of Tim and LARG biosensors, respectively. M.V., J.H. and G.D. developed and carried out computational analysis. G.D., J.S. and K.M.H. provided intellectual input in all phases of the study and directed the work. The paper was written by D.J.M., M.V., G.D., J.S. and K.M.H., with input from all authors.

Corresponding authors

Correspondence to Gaudenz Danuser, John Sondek or Klaus M. Hahn.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10.

Reporting Summary

Supplementary Video 1

Cdc42 (left) and Rac1 (right) activation reported by biosensors in MDA-MB-231 cells undergoing random edge motion.

Supplementary Video 2

Asef (left, middle) and Vav2 (right) activation reported by biosensors in MDA-MB-231 cells undergoing random edge motion.

Supplementary Video 3

Simultaneously imaged RhoGEF and Rho GTPase biosensors in MDA-MB-231 cells undergoing constitutive edge motion.

Supplementary Video 4

Simultaneously imaged Cdc42 and Rac1 biosensors in MDA-MB-231 cells undergoing constitutive edge motion.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Marston, D.J., Vilela, M., Huh, J. et al. Multiplexed GTPase and GEF biosensor imaging enables network connectivity analysis. Nat Chem Biol 16, 826–833 (2020).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing