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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.

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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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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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