Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

Abstract

Perception of ligands in the extracellular space by transmembrane receptors initiates signal transduction. The conformation change of the receptor induces changes of intracellular signalling components, including altered cellular concentration, altered subcellular location, altered conformation and altered interacting partners. Biochemical approaches have yielded a lot of information about these processes. However, methods that are compatible with analysis of single living cells are often preferred, since cells are highly organized and their response is usually spatially heterogeneous. In addition, the study of signalling cascades requires high temporal resolution. Fluorescence imaging approaches meet these requirements. Moreover, imaging approaches can be combined with genetically encoded green fluorescent protein-based probes that have a high selectivity and sensitivity for the process/molecule of interest. Nowadays, many genetically encoded probes are available for visualizing signalling in living cells. This review is centred on a key regulator of cellular signalling, protein kinase C (PKC). We will discuss imaging approaches that are used for analysing the molecules involved in activation of PKC, visualizing the dynamics of the location of PKC, measuring the conformation of PKC and quantifying the activity of PKC. These approaches are of general interest since they can be applied to study the dynamics, conformation and activity of any protein in living cells.

A Novel Neutralizing Antibody Specific to the DE Loop of VP1 Can Inhibit EV-D68 Infection in Mice

“…We next investigated if the great neutralization potency of A6-1 resulted in a high binding affinity to specific Ags VP1, VP1-1, VP1-2, and VP1-3 of the EV-D68 Fermon and KM strains using a FLIM/FRET assay (27,(34)(35)(36). FRET can be observed in the FLIM/FRET process; FRET activity shortens the lifetime of the donor fluorophore when an acceptor fluorophore exists because of energy transfer.…”

A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells

“…To solve this last issue, we developed a FRET biosensor of AURKA containing the full sequence of the kinase inserted between the donor–acceptor FRET pair. Similar approaches have previously been used to follow PRKGC and MELK, but the conformational changes observed by FRET were not directly assigned to kinase activation 54 55 . With the AURKA biosensor, we detected lifetime modifications in individual cells representing dynamic changes in the conformation of AURKA.…”

Chapter 24. Using Microfluidics, Real-time Imaging and Mathematical Modelling to study GPCR Signalling

“…Direct imaging of G protein dynamics has been achieved, 39 and with further development would be interesting to use for phaselocking analysis. Other relevant signalling components to image would be IP3, 52 and perhaps protein kinase C (PKC) 53 and the regulator of G protein signalling (RGS) proteins. 54 Microfluidic technology will also allow other stimulation patterns to be tested.…”