Single-molecule analysis has proven to be a powerful tool for the study of purified proteins in vitro (1 – 3). Single-molecule studies are not obscured by the ensemble- averaging inherent in conventional biochemical experiments. Direct observation of the dynamics and reactions of individual proteins gives us clear information about the molecular mechanisms of biological events. Recently, this technology has been extended to studies using living cells (4, 5) making it possible to quantify the dynamic and kinetic parameters of single molecule reactions in vivo.
Every protein in living cells works as part of a molecular network. As the components of these molecular networks and their construction are rapidly uncovered, one of the next objectives of cell biology is to quantitatively analyze the dynamic and kinetic parameters of the unitary processes of the networks and to understand how these unitary processes are integrated into the higher-order functions of networks. Single-molecule analysis in living cells will prove to be useful for this purpose since it provides several important advantages: (1) Single-molecule technique has the ultimate level of sensitivity and position detectability to observe reactions in cells. (2) Single-molecule measurements do not need synchronization of the reaction. This is a large advantage for the measurement of kinetic parameters of the intermediate steps of reaction networks. (3) Single-molecule measurements give information about the fluctuations and distributions of the dynamic and kinetic parameters. Considering the local heterogeneity of the structure and environment of living cells, the fluctuation and distribution of reactions are important to understand the mechanisms of cellular events. (4) Single-molecule analysis is a superior technique that allows quantification of the relationship between inputs and outputs of single events of protein reactions. These advantages of the single-molecule technique have already been successfully applied to the studies of various cellular events (6 – 11).
We are using single-molecule techniques in the studies of cellular response to a small peptide hormone, epidermal growth factor (EGF). In this review article, we will illustrate how single fluorescent molecules can be visualized in living cells and recent findings about the reactions of EGF receptor (EGFR) analyzed in single molecules.
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