Live‐cell super‐resolution imaging of PKA activity reveals microdomain organization and regulation

Abstract

Compartmentalized biochemical activities are crucial to many signaling processes in the cell. However, it has been difficult to visualize dynamic protein activities in living cells at the super‐resolution required to see their sub‐diffraction‐limit spatial regulation. Recently, we introduced a novel class of fluorescent biosensors that can detect biochemical activities in living cells at a resolution up to three‐fold better than the diffraction limit. Harnessing a specific binding‐induced change in protein fluorescence dynamics called Fluorescent fLuctuation INcrease by Contact (FLINC), these biosensors translate kinase activities or protein‐protein interactions into changes in fluorescence fluctuations. These fluctuations can then be quantified through Stochastic Optical Fluctuation Imaging (SOFI), permitting us to follow the protein activity dynamics, over tens of minutes, at super‐resolution. Utilizing one such biosensor for Protein Kinase A (PKA), we resolved minute activity microdomains on the plasma membrane of living cells, and discovered the role of clustered anchoring proteins in organizing these active PKA microdomains. We further demonstrate a new version of FLINC – REmoved Dronpa FLINC (red‐FLINC) – and use it to contextualize the PKA activity microdomains with the location of an additional protein. With red‐FLINC, live cell super‐resolution activity imaging requires only a single channel of the visible spectrum, freeing the others for imaging other proteins, or perturbing their activities with optogenetics. Using this technology. we determined the spatial relationship between membrane PKA activity microdomains and important participants in the PKA pathway such as AKAP79 and L‐type calcium channel. Our studies suggest that biochemical activities of the cell are spatially organized into an activity architecture.Support or Funding InformationNIH DP1 CA174423, R35 CA197622 and R01 DK073368This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.