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

Authors:
Publisher website
Google Scholar
Abstract
A quantitative sensor of spatial and temporal dynamics of activity of the protein tyrosine kinase Src shows that its activity peaks 1–2 μm from the leading edge of cells undergoing lamellipodial membrane extension, and the activation is correlated with protrusion velocity. 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.

This publication has 51 references indexed in Scilit: