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

Ananya Mukherjee, Randhir Singh, Sreeram Udayan, Sayan Biswas, Pothula Purushotham Reddy, Saumya Manmadhan, Geen George, Shilpa Kumar, Ranabir Das, Balaji M Rao, Akash Gulyani

eLife 2020 Feb 04

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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.

Citation

Ananya Mukherjee, Randhir Singh, Sreeram Udayan, Sayan Biswas, Pothula Purushotham Reddy, Saumya Manmadhan, Geen George, Shilpa Kumar, Ranabir Das, Balaji M Rao, Akash Gulyani. A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells. eLife. 2020 Feb 04;9