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

Akash Gulyani¹, Eric Vitriol, Richard Allen

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

Nature Chemical Biology

|June 14, 2011

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Researchers developed a new fluorescent biosensor using a fibronectin monobody scaffold. This versatile biosensor enables specific detection of targets like Src-family kinases (SFKs) in living cells with enhanced sensitivity.

Area of Science:

Cell Biology
Biochemistry
Molecular Biology

Background:

Existing fluorescent biosensors for live-cell imaging demand extensive optimization and specific designs for each target.
The utility of current biosensors is constrained by the limited availability of natural ligands with precise target specificity.

Purpose of the Study:

To engineer a generalizable scaffold for biosensor production using an engineered fibronectin monobody.
To develop a Src-family kinase (SFK) biosensor capable of binding tailored targets through high-throughput screening.
To create a biosensor that minimizes cell perturbation by sensing endogenous targets and enhancing sensitivity through direct dye excitation.

Main Methods:

Engineered a fibronectin monobody scaffold for target tailoring via high-throughput screening.

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Developed a Src-family kinase (SFK) biosensor by functionalizing a monobody specific for activated SFKs with a fluorescent dye.

Identified optimal dye attachment sites and modifications to prevent vesiculation in living cells.

Main Results:

The engineered monobody scaffold demonstrated generalizability for biosensor production.
The SFK biosensor exhibited increased fluorescence upon target binding.
Automated correlation revealed SFK activation specifically during cell protrusion, correlating with velocity and peaking near the leading edge.

Conclusions:

The fibronectin monobody scaffold offers a versatile platform for creating customized biosensors for various targets.
This approach enables sensitive detection of endogenous, unmodified targets in living cells with minimal perturbation.
The study provides new insights into the spatiotemporal dynamics of SFK activation during cell migration.