Many signal transduction pathways depend on a cascade of events that begins with the dimerization of cell surface receptors. In the case of epidermal growth factor (EGF) receptor, binding of EGF and receptor dimerization is followed by a conformational change in the receptor that results in autophosphorylation and activation of the receptor tyrosine kinase. While biochemical experiments, such as crosslinking and immunoprecipitation, have been used to study receptor dimerization, these techniques are limited by the fact that they require disruption of the cell. More recently, methods such as fluorescence resonance energy transfer (FRET) and complementation of β-galactosidase have opened the way to studying protein interactions in living cells. Now a report in the March issue of Nature Cell Biology (2, 168–172) shows how single molecule imaging technology can be used to watch the movements and interactions of individual EGF receptor molecules on the surface of cells.

The fluorescent dye Cy3 was conjugated to EGF and added to living human cells. Within one minute, two classes of fluorescent spots appeared at the cell surface (Figure). The fluorescence intensity of the minor class is about twice that of the major class. By monitoring the formation of spots, Yanagida and coworkers found that in most cases the fluorescence intensity of a spot suddenly increased by a factor of two. They interpreted the spontaneous doubling of fluorescence as the binding of a second molecule of EGF to a preformed receptor dimer complexed with a single EGF molecule. Using a monoclonal antibody that preferentially recognizes the phosphorylated (activated) form of the EGF receptor, they show that EGF receptor dimerization precedes the autophosphorylation of the receptor. Thus, they conclude that receptor dimerization occurs before receptor activation and signal transduction and that the receptor dimer bound to EGF is formed before a second molecule of EGF binds.

These types of single molecule experiments in living cells allow one to monitor individual members of a heterogeneous population and to identify and quantitatively compare the different subpopulations. Moreover, individual molecules can be studied under physiological conditions and monitored over time in a way that is difficult or impossible to do in standard experiments that yield information only on the average properties of a mixed population. By combining this exciting new method with manipulation techniques such as atomic force microscopy, and optical and magnetic tweezers, it should be possible to study the dynamic properties of more and more complicated biological systems in the future.