FRET measurements between small numbers of molecules identifies subtle changes in receptor interactions

Overexpression of HER2 alters the cellular behavior of EGF receptor (EGFR) and itself, with great implications on cell fate. To understand the molecular interactions underlying these alterations, we quantified the association between the two receptors by looking at efficiency changes in fluorescence resonance energy transfer (FRET) between a small number of molecules at the membrane of living cells. Human mammary epithelial (HME) cells expressing varying degrees of HER2 were studied, to identify and compare the degree of receptors interactions as a function of HER2 overexpression. A high resolution wide-field laser microscope combined with a high sensitivity cooled CCD camera was used to capture simultaneously donor and acceptor emissions. Alternating between green and red lasers every 80 msec, donor, FRET, and acceptor images were acquired and were used to calculate FRET efficiency. Automated image analysis was developed to create FRET efficiency maps from overlapping donor, acceptor and FRET images, and derive FRET efficiency histograms to quantify receptor-receptor interactions pixel by pixel. This approach enabled us to detect subtle changes in the average distance between EGFR molecules, and between EGFR and HER2. We found pre-existing EGFR homoassociations, and EGFR-HER2 heteroassociations in cells overexpressing HER2, and identified the changes in these interactions with ligand stimulation. These observations demonstrate the power of FRET measurements between small numbers of molecules in identifying subtle changes in molecular interactions in living cell.