Spatially regulated cell signaling is fundamental to virtually all biological processes. Genetically encodable biosensors are powerful tools that allow us to visualize the spatial compartmentalization of these signaling events in real time. Despite their utility, existing biosensors remain limited in both multiplexing and deep tissue imaging capabilities. In this study, we engineered chemigenetic fluorescent biosensors with reporting units employing a circularly-permuted HaloTag7 self-labeling protein tag. We used these chemigenetic biosensors to image spatially-resolved protein kinase A (PKA) activity in far red imaging windows on several spatial scales, achieving up to 1,250% dynamic range and drastically outperforming any other far-red PKA sensors currently available. We then showed that this chemigenetic biosensor design is generalizable to visualize the activity of other protein kinases. Notably, newly engineered substrate-based chemigenetic Ca2+/calmodulin-dependent protein kinase II (CaMKII) biosensors achieved a nearly 30-fold change upon stimulation, exhibiting the largest dynamic range and sensitivity for any kinase biosensor to date. Thus, these chemigenetic biosensors open up the door to highly sensitive interrogation of spatially and temporally regulated cell signaling across scales.

The video of this presentation is available here.