title: A biosensor generated via high-throughput screening quantifies cell edge Src dynamics.
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.
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Akash
authors.initials: A
authors.lastname: Gulyani
authors.forename: Eric
authors.initials: E
authors.lastname: Vitriol
authors.forename: Richard
authors.initials: R
authors.lastname: Allen
authors.forename: Jianrong
authors.initials: J
authors.lastname: Wu
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Gremyachinskiy
authors.forename: Steven
authors.initials: S
authors.lastname: Lewis
authors.forename: Brian
authors.initials: B
authors.lastname: Dewar
authors.forename: Lee M
authors.initials: LM
authors.lastname: Graves
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
authors.forename: Tim
authors.initials: T
authors.lastname: Elston
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Hahn
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D003198:Computer Simulation; D005353:Fibronectins / Q000378:metabolism*; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays / Q000379:methods*; D042541:Intracellular Space / Q000201:enzymology; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D011485:Protein Binding; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D005353:Fibronectins; D005456:Fluorescent Dyes; D019061:src-Family Kinases