Optogenetic imaging of protein activity in the synapse using 2-Photon fluorescence lifetime imaging microscopy

Abstract

Elucidating the spatiotemporal dynamics of proteins in living cells, including protein–protein interactions and conformational changes, is essential for understanding the mechanisms of intracellular signal transduction and cellular functions such as synaptic plasticity, cell motility, and cell division. One of the best ways to visualize protein activity with high spatiotemporal resolution is to utilize optogenetic probes, such as green fluorescent protein, in combination with fluorescence resonance energy transfer (FRET) techniques, which enable us to measure the distance between donor and acceptor fluorescent proteins fused to the signaling proteins. Among the various FRET detection methods, 2-photon fluorescence lifetime imaging microscopy (2pFLIM) is the ideal method to monitor FRET in subcellular compartments of living cells located deep within tissues, such as in brain slices. This review introduces the principle of the 2pFLIM-FRET for monitoring intracellular protein activities and protein–protein interactions using two examples: detecting small GTPase activity and monitoring actin polymerization in dendrites and synapses of hippocampal neurons in brain slices.