Optogenetic Imaging of Protein Activity Using Two-Photon Fluorescence Lifetime Imaging Microscopy.

Abstract

Spatiotemporal dynamics of cellular proteins, including protein-protein interactions and conformational changes, is essential for understanding cellular functions such as synaptic plasticity, cell motility, and cell division. One of the best ways to understand the mechanisms of signal transduction is to visualize protein activity with high spatiotemporal resolution in living cells within tissues. Optogenetic probes such as fluorescent proteins, in combination with Förster Resonance Energy Transfer (FRET) techniques, enable the measurement of protein-protein interactions and conformational changes in response to signaling events in living cells. Of the various FRET detection systems, two-photon fluorescence lifetime imaging microscopy (2pFLIM) is one of the methods best suited to monitoring FRET in subcellular compartments of living cells located deep within tissues, such as brain slices. This review will introduce the principle of 2pFLIM-FRET and the use of chromoproteins for imaging intracellular protein activities and protein-protein interactions. Also, we will discuss two examples of 2pFLIM-FRET application: imaging actin polymerization in synapses of hippocampal neurons in brain sections and detecting small GTPase Cdc42 activity in astrocytes.