The Fluorescent Two-Hybrid (F2H) Assay for Direct Analysis of Protein–Protein Interactions in Living Cells

Abstract

Information about protein interactions is crucial for the understanding of cellular processes. Current methods for the investigation of protein–protein interactions (PPIs) require either removal of the proteins from their normal cellular environment, perturbation of the cells or costly instrumentation and advanced technical expertise (Fields and Song, Nature 340:245–246, 1989; Deane et al., Mol Cell Proteomics 1:349–356, 2002; Kerppola, Nat Rev Mol Cell Biol 7:449–456, 2006; Blanchard et al., Mol Cell Proteomics 5:2175–2184, 2006; Miller et al., Mol Cell Proteomics 6:1027–1038, 2007; Miyawaki, Dev Cell 4:295–305, 2003; Parrish et al., Curr Opin Biotechnol 17:387–393, 2006; Sekar and Periasamy, J Cell Biol 160:629–633, 2003). Here, we describe a simple assay to directly visualize and analyze PPIs in single living cells. By adapting a lac operator/repressor system, we generated a stable nuclear interaction platform. A fluorescent bait protein is tethered to the interaction platform and assayed for co-localization of fluorescent prey fusion proteins. This fluorescent two-hybrid (F2H) assay allows the investigation of cell cycle dependent PPIs. With this cell based assay protein interactions even from different subcellular compartments can be visualized in real time (Zolghadr et al., Mol Cell Proteomics 7:2279–2287, 2008). The simple optical readout enables automated imaging systems to segment and analyze the acquired data for high-throughput screening of PPIs in living cells in response to external stimuli and chemical compounds.