Genetically-Encoded Biosensors for Visualizing Live-cell Biochemical Activity at Superresolution

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with their compartmentalization. Here we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to three-fold better than the diffraction limit. Utilizing specific, binding-induced changes in protein fluorescence dynamics, these biosensors translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A Protein Kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membrane of living cells and uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture, whose structural and functional characteristics can be revealed by these new biosensors.