Towards Imaging the Dynamics of Protein Signalling

In living cells protein signalling is a complex dynamic process. We discuss the spatiotemporal aspects of such signalling and how the variety of different optical imaging techniques can deal with the necessary temporal and high optical resolution. We consider techniques like confocal laser scanning microscopy, kilobeam scanning, nonlinear microscopy, selective plane imaging, structured illumination, total internal reflection fluorescence microscopy, near-field optical microscopy and fluorescence lifetime imaging. Complementary to the technological aspects we discuss different approaches of how to make specific proteins visible, including bioluminescence, autofluorescence, immunohistochemistry, fluorescence-fusion proteins and specific covalent labelling of proteins. Finally we introduce concepts to image protein signalling. Protein dynamics are considered on the basis of translocations of fluorescent protein tagged conventional protein kinase C. Protein–protein interactions can best be investigated in living cells by molecular interaction of fluorescence-protein tags, namely bimolecular fluorescence complementation or Förster resonance energy transfer (FRET). Today, modern imaging techniques even allow the investigation of biochemical reactions such as kinasemediated phosphorylation in living cells. Such genetically engineered biosensors make use of conformational changes that induce changes of intramolecular FRET.