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

ABSTRACT: 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.

SUBMITTER: Mukherjee A

PROVIDER: S-EPMC7000222 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Mukherjee Ananya A Singh Randhir R Udayan Sreeram S Biswas Sayan S Reddy Pothula Purushotham PP Manmadhan Saumya S George Geen G Kumar Shilpa S Das Ranabir R Rao Balaji M BM Gulyani Akash A

eLife 20200204

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) analys ...[more]

PMID: 32017701

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Project description:SignificanceUnderstanding how the valveless embryonic heart pumps blood is essential to elucidate biomechanical cues regulating cardiogenesis, which is important for the advancement of congenital heart defects research. However, methods capable of embryonic cardiac pumping analysis remain limited, and assessing this highly dynamic process in mammalian embryos is challenging. New approaches are critically needed to address this hurdle.AimWe report an imaging-based approach for functional assessment of localized pumping dynamics in the early tubular embryonic mouse heart.ApproachFour-dimensional optical coherence tomography was used to obtain structural and Doppler hemodynamic imaging of the beating heart in live mouse embryos at embryonic day 9.25. The pumping assessment was performed based on the volumetric blood flow rate, flow resistance within the heart tube, and pressure gradient induced by heart wall movements. The relation between the blood flow, the pressure gradient, and the resistance to flow were evaluated through temporal analyses and Granger causality test.ResultsIn the ventricles, our method revealed connections between the temporal profiles of pressure gradient and volumetric blood flow rate. Statistically significant causal relation from the pressure gradient to the blood flow was demonstrated. Our analysis also suggests that cardiac pumping in the early ventricles is a combination of suction and pushing. In contrast, in the outflow tract, where the conduction wave is slower than the blood flow, we did not find significant causal relation from pressure to flow, suggesting that, different from ventricular regions, the local active contraction of the outflow tract is unlikely to drive the flow in that region.ConclusionsWe present an imaging-based approach that enables localized assessment of pumping dynamics in the mouse tubular embryonic heart. This method creates a new opportunity for functional analysis of the pumping mechanism underlying the developing mammalian heart at early stages and could be useful for studying biomechanical changes in mutant embryonic hearts that model congenital heart defects.

Dataset Information

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

Publications

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

Similar Datasets

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