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

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Nature Chemical Biology (Impact Factor: 13). 06/2011; 7(7):437-44. DOI: 10.1038/nchembio.585
Source: PubMed


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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Available from: Klaus M Hahn, Sep 01, 2014
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    • "fy endoge - nous factors with recombinant binders is based on solvatochro - matic fluorescent dyes whose fluorescence increases upon antigen binding ( Toutchkine et al . , 2003 ; Nalbant et al . , 2004 ) . Site - specific attachment of these dyes to a Src - specific mono - body for example allowed the quantitation of Src dynamics in living cells ( Gulyani et al . , 2011 ) . So far , however , this approach requires in vitro dye conjugation and intracellular application via microinjection , which limits its utility in cell biology . In general , recombinant SNAP - / Halo - / CLIP - tag techniques ( Kep - pler et al . , 2003 ; Gautier et al . , 2008 ; Los et al . , 2008 ) rely on self - labeling protein "
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    • "A particularly elegant application of FN3 domain binders as fluorescent biosensors was recently reported by Gulyani et al. (Gulyani et al., 2011) A Src-family kinase (SFK) biosensor was prepared using a FN3 domain specific for activated SFKs. Binding of the FN3 domain to its target was monitored by attachment of a bright, environmentally sensitive fluorescent dye to the scaffold protein where target protein binding was shown to increase fluorescence. "

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