2020 journal article

A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells

ELIFE, 9.

By: A. Mukherjee *, R. Singh *, S. Udayan*, S. Biswas *, P. Reddy*, S. Manmadhan*, G. George*, S. Kumar* ...

MeSH headings : Animals; Biosensing Techniques; Cell Line; Cell Physiological Phenomena / genetics; Fluorescence Resonance Energy Transfer; Fluorescent Dyes / metabolism; HEK293 Cells; Humans; Magnetic Resonance Spectroscopy; Mice; Phosphorylation / genetics; Proto-Oncogene Proteins c-fyn / chemistry; Proto-Oncogene Proteins c-fyn / genetics; Proto-Oncogene Proteins c-fyn / metabolism; Signal Transduction / genetics; Yeasts / genetics
TL;DR: A sensitive, minimally-perturbing fluorescence-resonance-energy-transfer biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling and clarifies how compartmentalized Src-kinase activity may drive cell fate. (via Semantic Scholar)
UN Sustainable Development Goal Categories
Source: Web Of Science
Added: February 27, 2020

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.