Real-Time Protein-Fragment Complementation Assays for Studying Temporal, Spatial, and Spatiotemporal Dynamics of Protein-Protein Interactions in Living Cells.

Abstract

Here, we present detailed protocols for direct, real-time protein-fragment complementation assays (PCAs) for studying the spatiotemporal dynamics of protein-protein interactions (PPIs). The assays require the use of two fluorescent reporter proteins-the "Venus" version of yellow fluorescent protein (vYFP), and the monomeric infrared fluorescent protein 1.4 (IFP 1.4)-or two luciferase reporter proteins-Renilla (Rluc) and Gaussia (Gluc). The luciferase PCAs can be used to study the temporal dynamics of PPIs in any cellular compartment and on membranes. The full reversibility of these PCAs assures accurate measurements of the kinetics of PPI assembly/disassembly for processes that occur anywhere in a living cell and over time frames of seconds to hours. vYFP PCA, and all PCAs based on green fluorescent protein and its variants, are irreversible and can be used to trap and visualize rare and transient complexes and follow dynamic relocalization of constitutive complexes. vYFP PCA is limited in that accurate measurements of temporal changes in PPIs are not possible owing to the slow maturation time of vYFP (minutes to hours) and the irreversibility of its PCA that traps the complexes, thereby preventing the dissociation of PPIs that, in some instances, might cause spurious mislocalization of protein complexes. The limitations of vYFP PCA are overcome with IFP PCA, which is fully reversible and thus can be used to study spatiotemporal dynamics of PPIs on the timescale of seconds. All of these PCAs are sensitive enough to detect interactions among proteins expressed at endogenous levels in vivo.