Reporter of protein behaviour
As a genetically encodable fluorescent tag, GFP has been used to monitor a variety of protein behaviors:
Dynamic localization of proteins
As sequencing of the human genome nears completion, increasing emphasis is being placed on the elucidation of protein function. Understanding the dynamic intracellular distribution of a protein can be the first clue toward unraveling its function. GFP is proving to be an invaluable tool in this effort, since it allows scientists to visualize the protein in real-time in a living cell or organism.
Protein translocation
A burgeoning application area for GFP involves tracking the movement of proteins within or between cells in response to extra cellular stimuli. The movement of a target protein in vivo may be rapid and transient, and therefore difficult or even impossible to detect in a traditional fixed-cell assay. With the aid of high-speed, live-cell fluorescence imagers such as the IN Cell Analyzer, previously intractable targets can be measured by quantifying the real-time redistribution of GFP-tagged proteins after treatment with test compounds. Live cellular translocation assays are becoming increasingly attractive in drug discovery for the development of pharmaceutical screens.
Protein turnover
Protein expression and degradation are exquisitely timed and tightly regulated processes. GFP fusion proteins have been used to elucidate the kinetics and regulation of protein turnover.
Changes in cell and organelle morphology
GFP fusion proteins targeted to particular regions of the cell can be used as dynamic markers of general cell shape or organelle morphology. Agonist-induced changes in morphology can be quantified by image analysis. GFP reporters can monitor shape changes indicative of important cellular processes such as apoptosis (regulated cell death), proliferation, adhesion, migration and neurite outgrowth.
Protein-protein interactions
Fluorescence resonance energy transfer (FRET) has been successful in detecting interactions between molecules tagged with fluorescent proteins whose excitation and emission spectra overlap. Examples of GFP-based FRET applications include assays to detect protease activity, transcription factor dimerization and calcium changes.
R N Day et al. Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 2001, 25(1):4-8.
Changes in local pH
A number of GFP variants have been optimized for use as pH sensors. For example, Miesenbock et al., (Nature 394:192-195) have generated pH-sensitive GFP with excitation profiles tailored to respond to pH changes occurring along exocytic and endocytic pathways. Such pH-sensitive GFP could prove useful in monitoring processes such as synaptic transmission and regulated secretion.
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