Three-Fragment Fluorescence Complementation Coupled with Photoactivated Localization Microscopy for Nanoscale Imaging of Ternary Complexes
ABSTRACT:Many cellular processes are governed by molecular machineries that involve multiple protein interactions. However, visualizing and identifying multiprotein complexes such as ternary complexes inside cells is always challenging, particularly in the subdiffraction cellular space. Here, we developed a three-fragment fluorescence complementation system (TFFC) based on the splitting of a photoactivatable fluorescent protein, mIrisFP, for the imaging of ternary complexes inside living cells. Using a combination of TFFC and photoactivated localization microscopy (PALM), namely, the TFFC-PALM technique, we are able to identify the multi-interaction of a ternary complex with nanometer-level spatial resolution and single-molecule sensitivity. The TFFC-PALM system has been further applied to the analysis of the Gs ternary complex, which is composed of αs, β1, and γ2 subunits, providing further insights into the subcellular localization and function of G protein subunits at the single-molecule level. The TFFC-PALM represents a valuable method for the visualization and identification of ternary complexes inside cells at the nanometer scale.
