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

• DOI: 10.7554/elife.50571
• Publication Date: 2020-02-04T15:00:16Z
• Authors: Ananya Mukherjee; Randhir Singh; Sreeram Udayan; Sayan Biswas; Pothula Purushoth...; Saumya Manmadhan; Geen George; Shilpa Kumar; Ranabir Das; Balaji M Rao; Akash Gulyani
• Keywords: FYN; Crosstalk; Src family kinase

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 SFK-Fyn live cells. We deployed combinatorial library screening to isolate binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis F29 specificity Fyn over homologous SFKs. Using F29, engineered sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) (FynSensor) that reveals cellular be spatially localized, pulsatile sensitive adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced pre-activated intracellular zones. However, inhibition focal-adhesion-kinase not only attenuates activity, but abolishes growth-factor modulation. FynSensor imaging uncovers organized, sensitized signaling clusters, direct crosstalk between integrin growth-factor-signaling, clarifies how compartmentalized Src-kinase may drive fate.