Fluorescent Protein-Based Biosensors

Marina Polito, Pierre Vincent, Elvire Guiot, Polito, Marina, Vincent, Pierre, Guiot, Elvire

Cyclic-AMP dependent protein kinase (PKA) is present in most branches of the animal kingdom, and is an example in the nervous system where a kinase effector integrates the cellular effects of various neuromodulators. The recent development of FRET-based biosensors, such as AKAR, now allows the direct measurement of PKA activation in living cells by simply measuring the ratio between the fluorescence emission at the CFP and YFP wavelengths upon CFP excitation. This novel approach provides data with a temporal resolution of a few seconds at the cellular and even subcellular level, opening a new avenue of understanding the integration processes in space and time. Our protocol has been optimized to study morphologically intact mature neurons and we describe how simple and cheap wide-field imaging, as well as more elaborate two-photon imaging, allows real-time monitoring of PKA activation in pyramidal cortical neurons in neonate rodent brain slices. In addition, many practical details presented here also pertain to image analysis in other cellular preparations, such as cultured cells. Finally, this protocol can also be applied to the various other CFP-YFP-based FRET biosensors that are available for other kinases or other intracellular signals. It is likely that this kind of approach will be generally applicable to a broad range of assays in the near future.