For most fluorescence imaging applications, the fluorescent label is a biologically inert participant that is used merely as a visible marker5. By the very nature of their barrel-like structures, which effectively shield the chromophore from the external environment, AFPs are well suited to these more passive applications. Typical passive uses of AFPs include monitoring the appearance, degradation, location or translocation of appropriate partner proteins to which they are fused.
Knowledge of protein localization can provide a wealth of information about a proteinís function, activation state and interactions with other molecules4. Given the compartmentalization of eukaryotic cells, a proteinís localization is typically related to its function and can therefore be an important step towards the full understanding of its physiological role. Ideally, localization information indicates not only where a protein is found, but when it is found there and whether it changes localization: in fact, changes in localization may result from cell signaling events, environmental changes and progression through the cell cycle4.
The most popular applications of fluorescent proteins5 involve exploiting them for imaging of the localization and dynamics of specific organelles or recombinant proteins in live cells. For imaging of a specific organelle, standard molecular biology techniques are used to fuse the gene encoding the fluorescent protein to a cDNA encoding a protein or peptide known to localize to that specific organelle. This fusion is done such that the chimeric gene will be expressed as a single polypeptide, creating a covalent link between the targeting motif and the fluorescent protein. A plasmid containing the chimeric gene under control of a suitable promoter is used to transfect mammalian cells that then express the gene to produce the corresponding chimeric protein. The chimera localizes to the target organelle and thus renders it fluorescent. Through the use of fluorescence microscopy, the morphology, dynamics, and distribution of the organelle can be imaged as a function of time. The procedure for imaging of a fusion between a fluorescent protein and a specific protein-of-interest (in order to gain insight into its localization and dynamics) is identical. The availability of a broad selection of colors of fluorescent proteins19 has provided researchers with the means to image the localization of multiple organelles and/or proteins-of-interest, simultaneously.
Fluorescence as a spatial marker: Localizing gene activity and transcripts. RNA localization can be visualized in live cells through fusion of AFP to an RNA-binding protein or domain5.
Fluorescence as a temporal marker: Analysing gene expression5.