Fluorescent probes of cell signaling.

Fluorescence has long been recognized as a powerful tool for probing biological structure and function. Because probe molecules can be very much more fluorescent than the constituents of most biological specimens, the signal for the exogenous fluorophores can be measured continuously and nondestructively with excellent spatial and temporal resolution in living cells (Waggoner 1 986). The earliest developed and most straight­ forward uses of fluorescent groups are simply as positional tags or markers. Examples are immunofluorescence labeling (Nairn 1 976), fluorescent analog cytochemistry (Taylor et al 1 986a), vital staining of organelles (Pagano & Sleight 1 985, Wang & Taylor 1988), assessment of cell mor­ phology or intercellular coupling with microinjected tracers (Stewart 1 98 1 ), measurement of distances between probes by fluorescence energy transfer (Stryer 1978, Uster & Pagano 1 986), and measurement of diffusion coefficients and exchange rates by photo bleaching recovery (Elson 1986). The common feature of such applications is that the main role of the fluorescent group is merely to signal its presence and location rather than to sense its environment. The main criteria for such fluorescent tags are simple: wavelengths of excitation and emission, brightness, photostability (Mathies & Stryer 1 986), size and charge. Therefore a few fluorophores (e.g. fluoresceins, rhodamines, naphthalimides, phycobiliproteins, nitro­ benzoxadiazole) tend to get used over and over, often attached by rather standardized techniques to different macromolecules. A second type of application of fluorescent probes involves attachment of the fluorophore to a purified macromolecule to sense conformational change of the latter (Y guerabide 1 972, Cooke 1982, Lakowicz 1 983). The