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A biosensor generated via high-throughput screening quantifies cell edge Src dynamics

Nature Chemical Biology volume 7, pages 437–444 (2011)Cite this article

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A Corrigendum to this article was published on 18 July 2012

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Abstract

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high-throughput screening. We made this Src-family kinase (SFK) biosensor by derivatizing a monobody specific for activated SFKs with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and changes to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFKs are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 μm from the leading edge.

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Figure 1: Screening an FN3 monobody library leads to a biosensor for Src family activity.
Figure 2: Fibronectin monobody 1F11 preferentially binds active Src.
Figure 3: Screening for responsive sensor variants, selecting dye and site for dye labeling.
Figure 4: Src activation dynamics in living cells.
Figure 5: Automated edge analysis and line scans reveal a distinct zone of SFK activity that is correlated with protrusion velocity.
Figure 6: Modeling of the 1F11 dye–SH3 interface.

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  • 15 June 2012

    In the version of this article initially published, the residue numbers indicated in the y-axis labels in Figure 3c were incorrectly written as 24, 53, 55 and 53 from the origin but should have read 24, 52, 53 and 55. The error has been corrected for the PDF and HTML versions of this article.

References

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

    Article  CAS  Google Scholar 

  2. Toutchkine, A., Kraynov, V. & Hahn, K. Solvent-sensitive dyes to report protein conformational changes in living cells. J. Am. Chem. Soc. 125, 4132–4145 (2003).

    Article  CAS  Google Scholar 

  3. Toutchkine, A., Nguyen, D.V. & Hahn, K.M. Merocyanine dyes with improved photostability. Org. Lett. 9, 2775–2777 (2007).

    Article  CAS  Google Scholar 

  4. Toutchkine, A., Nguyen, D.V. & Hahn, K.M. Simple one-pot preparation of water-soluble, cysteine-reactive cyanine and merocyanine dyes for biological imaging. Bioconjug. Chem. 18, 1344–1348 (2007).

    Article  CAS  Google Scholar 

  5. Koide, A., Bailey, C.W., Huang, X. & Koide, S. The fibronectin type III domain as a scaffold for novel binding proteins. J. Mol. Biol. 284, 1141–1151 (1998).

    Article  CAS  Google Scholar 

  6. Karatan, E. et al. Molecular recognition properties of FN3 monobodies that bind the Src SH3 domain. Chem. Biol. 11, 835–844 (2004).

    Article  CAS  Google Scholar 

  7. Koide, A., Abbatiello, S., Rothgery, L. & Koide, S. Probing protein conformational changes in living cells by using designer binding proteins: application to the estrogen receptor. Proc. Natl. Acad. Sci. USA 99, 1253–1258 (2002).

    Article  CAS  Google Scholar 

  8. Sidhu, S.S. & Koide, S. Phage display for engineering and analyzing protein interaction interfaces. Curr. Opin. Struct. Biol. 17, 481–487 (2007).

    Article  CAS  Google Scholar 

  9. Parsons, S.J. & Parsons, J.T. Src family kinases, key regulators of signal transduction. Oncogene 23, 7906–7909 (2004).

    Article  CAS  Google Scholar 

  10. Thomas, S.M. & Brugge, J.S. Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13, 513–609 (1997).

    Article  CAS  Google Scholar 

  11. Playford, M.P. & Schaller, M.D. The interplay between Src and integrins in normal and tumor biology. Oncogene 23, 7928–7946 (2004).

    Article  CAS  Google Scholar 

  12. Bromann, P.A., Korkaya, H. & Courtneidge, S.A. The interplay between Src family kinases and receptor tyrosine kinases. Oncogene 23, 7957–7968 (2004).

    Article  CAS  Google Scholar 

  13. 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 

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

    Article  CAS  Google Scholar 

  15. Ting, A.Y., Kain, K.H., Klemke, R.L. & Tsien, R.Y. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl. Acad. Sci. USA 98, 15003–15008 (2001).

    Article  CAS  Google Scholar 

  16. Cowan-Jacob, S.W. et al. The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. Structure 13, 861–871 (2005).

    Article  CAS  Google Scholar 

  17. Gardner, O.S., Dewar, B.J., Earp, H.S., Samet, J.M. & Graves, L.M. Dependence of peroxisome proliferator-activated receptor ligand-induced mitogen-activated protein kinase signaling on epidermal growth factor receptor transactivation. J. Biol. Chem. 278, 46261–46269 (2003).

    Article  CAS  Google Scholar 

  18. Dewar, B.J. et al. Capacitative calcium entry contributes to the differential transactivation of the epidermal growth factor receptor in response to thiazolidinediones. Mol. Pharmacol. 72, 1146–1156 (2007).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. 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 

  21. 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 

  22. Glick, D., Barth, S. & Macleod, K.F. Autophagy: cellular and molecular mechanisms. J. Pathol. 221, 3–12 (2010).

    Article  CAS  Google Scholar 

  23. Reis, R.C., Sorgine, M.H. & Coelho-Sampaio, T. A novel methodology for the investigation of intracellular proteolytic processing in intact cells. Eur. J. Cell Biol. 75, 192–197 (1998).

    Article  CAS  Google Scholar 

  24. Panchuk-Voloshina, N. et al. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47, 1179–1188 (1999).

    Article  CAS  Google Scholar 

  25. Hodgson, L., Shen, F. & Hahn, K. Biosensors for characterizing the dynamics of rho family GTPases in living cells. Curr. Protoc. Cell Biol. 46, 14.11.1–14.11.26 (2010).

    Google Scholar 

  26. Bright, G.R., Fisher, G.W., Rogowska, J. & Taylor, D.L. Fluorescence ratio imaging microscopy. Methods Cell Biol. 30, 157–192 (1989).

    Article  CAS  Google Scholar 

  27. Buccione, R., Orth, J.D. & McNiven, M.A. Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat. Rev. Mol. Cell Biol. 5, 647–657 (2004).

    Article  CAS  Google Scholar 

  28. Veracini, L. et al. Two functionally distinct pools of Src kinases for PDGF receptor signalling. Biochem. Soc. Trans. 33, 1313–1315 (2005).

    Article  CAS  Google Scholar 

  29. Veracini, L. et al. Two distinct pools of Src family tyrosine kinases regulate PDGF-induced DNA synthesis and actin dorsal ruffles. J. Cell Sci. 119, 2921–2934 (2006).

    Article  CAS  Google Scholar 

  30. Sandilands, E., Brunton, V.G. & Frame, M.C. The membrane targeting and spatial activation of Src, Yes and Fyn is influenced by palmitoylation and distinct RhoB/RhoD endosome requirements. J. Cell Sci. 120, 2555–2564 (2007).

    Article  CAS  Google Scholar 

  31. Sandilands, E. et al. RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane. Dev. Cell 7, 855–869 (2004).

    Article  CAS  Google Scholar 

  32. Cary, L.A., Klinghoffer, R.A., Sachsenmaier, C. & Cooper, J.A. SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading. Mol. Cell. Biol. 22, 2427–2440 (2002).

    Article  CAS  Google Scholar 

  33. Fincham, V.J. & Frame, M.C. The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility. EMBO J. 17, 81–92 (1998).

    Article  CAS  Google Scholar 

  34. Frame, M.C., Fincham, V.J., Carragher, N.O. & Wyke, J.A. v-Src's hold over actin and cell adhesions. Nat. Rev. Mol. Cell Biol. 3, 233–245 (2002).

    Article  CAS  Google Scholar 

  35. Machacek, M. & Danuser, G. Morphodynamic profiling of protrusion phenotypes. Biophys. J. 90, 1439–1452 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Renard, M. et al. Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies. J. Mol. Biol. 318, 429–442 (2002).

    Article  CAS  Google Scholar 

  38. Koide, A., Jordan, M.R., Horner, S.R., Batori, V. & Koide, S. Stabilization of a fibronectin type III domain by the removal of unfavorable electrostatic interactions on the protein surface. Biochemistry 40, 10326–10333 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  40. Martinez-Quiles, N., Ho, H.Y., Kirschner, M.W., Ramesh, N. & Geha, R.S. Erk/Src phosphorylation of cortactin acts as a switch on-switch off mechanism that controls its ability to activate N-WASP. Mol. Cell. Biol. 24, 5269–5280 (2004).

    Article  CAS  Google Scholar 

  41. Cory, G.O., Garg, R., Cramer, R. & Ridley, A.J. Phosphorylation of tyrosine 291 enhances the ability of WASp to stimulate actin polymerization and filopodium formation. Wiskott-Aldrich Syndrome protein. J. Biol. Chem. 277, 45115–45121 (2002).

    Article  CAS  Google Scholar 

  42. Suetsugu, S. et al. Sustained activation of N-WASP through phosphorylation is essential for neurite extension. Dev. Cell 3, 645–658 (2002).

    Article  CAS  Google Scholar 

  43. Plattner, R., Kadlec, L., DeMali, K.A., Kazlauskas, A. & Pendergast, A.M. c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF. Genes Dev. 13, 2400–2411 (1999).

    Article  CAS  Google Scholar 

  44. Sini, P., Cannas, A., Koleske, A.J., Di Fiore, P.P. & Scita, G. Abl-dependent tyrosine phosphorylation of Sos-1 mediates growth-factor-induced Rac activation. Nat. Cell Biol. 6, 268–274 (2004).

    Article  CAS  Google Scholar 

  45. Yang, L., Kowalski, J.R., Zhan, X., Thomas, S.M. & Luscinskas, F.W. Endothelial cell cortactin phosphorylation by Src contributes to polymorphonuclear leukocyte transmigration in vitro. Circ. Res. 98, 394–402 (2006).

    Article  CAS  Google Scholar 

  46. Bouton, A.H., Riggins, R.B. & Bruce-Staskal, P.J. Functions of the adapter protein Cas: signal convergence and the determination of cellular responses. Oncogene 20, 6448–6458 (2001).

    Article  CAS  Google Scholar 

  47. Polte, T.R. & Hanks, S.K. Complexes of focal adhesion kinase (FAK) and Crk-associated substrate (p130(Cas)) are elevated in cytoskeleton-associated fractions following adhesion and Src transformation. Requirements for Src kinase activity and FAK proline-rich motifs. J. Biol. Chem. 272, 5501–5509 (1997).

    Article  CAS  Google Scholar 

  48. DerMardirossian, C., Rocklin, G., Seo, J.Y. & Bokoch, G.M. Phosphorylation of RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling. Mol. Biol. Cell 17, 4760–4768 (2006).

    Article  CAS  Google Scholar 

  49. Timpson, P., Jones, G.E., Frame, M.C. & Brunton, V.G. Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity. Curr. Biol. 11, 1836–1846 (2001).

    Article  CAS  Google Scholar 

  50. 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).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C. MacNevin for assistance with dye synthesis, F. Shen for help with imaging studies, D. Renfrew for assistance with computational modeling, A. Nguyen for technical assistance and B. Clarke for administrative assistance. We gratefully acknowledge funding from the American Heart Association (A.G.) and the US National Institutes of Health (GM GM082288 and GM057464 to K.M.H.).

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  1. Akash Gulyani and Eric Vitriol: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Akash Gulyani, Eric Vitriol, Richard Allen, Jianrong Wu, Dmitriy Gremyachinskiy, Brian Dewar, Lee M Graves, Tim Elston & Klaus M Hahn

  2. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Steven Lewis & Brian Kuhlman

  3. Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

    Brian K Kay

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Contributions

A.G. developed the biosensors, with help from B.D. and L.M.G. on phosphorylation assays. E.V. carried out live-cell imaging studies, assisted by A.G. R.A. and T.E. developed the image analysis algorithms and carried out automated analysis of SFK activity, with help from J.W. S.L. and B.K. modeled dye-protein interactions. D.G. synthesized the dyes. B.K.K. provided protein constructs and input regarding FN3 screening and structure. K.M.H. conceived the study, directed the research and wrote the final manuscript based on contributions from all authors.

Corresponding author

Correspondence to Klaus M Hahn.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Methods (PDF 1299 kb)

Supplementary Video 1

Src activation in fibroblasts with PDGF stimulation (AVI 5283 kb)

Supplementary Video 2

Src activation at the cell edge in PDGF stimulated fibroblasts (AVI 482 kb)

Supplementary Video 3

Src activation in randomly migrating epithelial cells (AVI 5024 kb)

Supplementary Video 4

Src activation at the cell edge and the effect of inhibitor (AVI 865 kb)

Supplementary Video 5

Merobody sensor is sensitive to Src activity. (AVI 4690 kb)

Supplementary Video 6

Src merobody sensor localization at the cell edge is dependent on Src activity (AVI 649 kb)

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Gulyani, A., Vitriol, E., Allen, R. et al. A biosensor generated via high-throughput screening quantifies cell edge Src dynamics. Nat Chem Biol 7, 437–444 (2011). https://doi.org/10.1038/nchembio.585

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