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Until recently, studies in this field of signal transduction have involved the "what" and "when" of signaling. Who talks to whom and for how long? With the advent of genetically encoded fluorescent proteins, it has become possible to monitor signaling events in living cells in real time. This has added the dimension of "where" to the study of cellular signaling. This lecture, which is a part of "Cell Signaling Systems: A Course for Graduate Students," provides a survey of how green fluorescent protein (GFP)-tagged probes for signaling events have been used to elucidate new pathways, to describe the kinetics of signaling events at the single-cell level, and to reveal upon which subcellular compartments these events take place. Some of the findings confirm previous ones using biochemical techniques, and others have been surprising. Examples include those utilizing protein localization, relocalization, fluorescence recovery after photobleaching (FRAP), and fluorescence resonance energy transfer (FRET). The design of FRET probes is described. The detection of small guanosine triphosphatase (GTPase) signaling in living cells is used as an example to explore the creative and diverse ways investigators have developed to look at this system.
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