TitleA Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells.
Publication TypeJournal Article
Year of Publication2020
AuthorsMukherjee A, Singh R, Udayan S, Biswas S, Reddy PPurushotha, Manmadhan S, George G, Kumar S, Das R, Rao BM, Gulyani A
Date Published2020 Feb 04

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 () 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. 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.

Alternate JournalElife
PubMed ID32017701
PubMed Central IDPMC7000222
Grant ListJRF-SRF Fellowship / / University Grants Commission /
DBT-RA Program in Biotechnology and Life Sciences / / Department of Biotechnology, Ministry of Science and Technology /
Grant BT/PR8071/MED/32/290/2013 / / Department of Biotechnology, Ministry of Science and Technology /
BT/IN/BMBF/03/AG/2015-16 / / Department of Biotechnology, Ministry of Science and Technology /
BT/IN/Denmark/07/RSM/2015-2016 / / Department of Biotechnology, Ministry of Science and Technology /