Bright, sensitive fluorescent probes that respond to changes in the cellular microenvironment are extremely valuable for imaging cellular dynamics. We report a simple, one-step synthesis of a new hemicaynine (HC-1) dye as a sensitive, redemitting (l max -610 nm) fluorogenic probe for micro-viscosity and local order in diverse environments, including live cells. HC-1 responds to increasing micro-viscosity through changes in fluorescence intensity and lifetime, and is sensitive enough to report dynamic micellar self-assembly. While HC-1 shows properties of a molecular 'rotor', time-dependent density functional theoretical analysis reveals that in HC-1, an inhibition of photo-isomerization in viscous environment is the likely cause of fluorescence enhancement. HC-1 localizes to mitochondria in live cells and responds to mitochondrial ordering through a significant increase in fluorescence. Strikingly, we show that HC-1 is also a sensitive probe for the spatial heterogeneity of mitochondrial organization in embryonic stem cells as well as dynamic remodeling of the mitochondria in early-differentiated cells.
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.
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