Measuring protein interactions using Forster resonance energy transfer and fluorescence lifetime imaging microscopy

The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Forster resonance energy transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBP alpha). C/EBP alpha is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1 alpha). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBP alpha. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBP alpha is sufficient for the interaction with HP1 alpha in regions of heterochromatin. (C) 2013 Elsevier Inc. All rights reserved.