Genomics and proteomics approaches provide insight into protein functions and enable quantification of protein levels under different cellular conditions, but they are incapable of addressing when and where a protein is active3. Yet protein localization and protein function are intricately linked3,4. Moreover, compartmentalized signaling reactions may lead to different cellular phenotypes. Thus, imaging protein activity and the cellular cues that stimulate activity in living cells can provide an important complement to classical genomic and proteomic approaches.
We are witnessing the rapid development of biosensors based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its siblings from other organisms and engineered variants of members of the GFP-family of proteins2. The advantage of genetically encoded fluorescent probes is that they enable the researcher to study a specific protein or cellular signal within the complex environment of the living cell3. This approach preserves the spatial and temporal control of protein function and signaling cascades. Thus, the researcher can assess when and where a protein is active in a cell, as well as identify how a stimulus influences the dynamics of signaling cues, which in turn dictate protein function3. Moreover, fluorescent probes can be easily incorporated into cells, tissues, and even organisms. Once these probes are loaded, they permit long-term imaging over days or longer (in the case of stable incorporation)3 and they very rarely cause photodynamic toxicity5.
From now on we refer to the entire class of Aequorea-derived fluorescent proteins as AFPs.