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The development of photoactivatable fluorescent proteins began in the mid 1990s and finally yielded truly useful tools in 2002. Exposure of these proteins to light of a certain wavelength and intensity changes their fluorescence, allowing researchers to highlight proteins in discrete regions of cells and track their movement directly rather than watching the movement of proteins into a bleached space as was previously done. A report by Lukyanov and colleagues in the November issue of Nature Biotechnology describes the creation of a new dual-color photoactivatable monomeric fluorescent protein they call photoswitchable cyan fluorescent protein, or PS-CFP, that promises to further simplify protein tracking experiments (Chudakov et al., 2004).

PS-CFP represents the next generation of optical highlighter molecules and has some distinct advantages over the currently published proteins. It offers a higher contrast between pre- and postactivation fluorescence than either PA-GFP (Patterson and Lippincott-Schwartz, 2002) or KFP1 (Chudakov et al., 2003). This contrast is on the same scale as that for Kaede (Ando et al., 2002), but the protein is a monomer instead of an obligate tetramer. Following exposure to relatively intense 405-nm light, this cyan fluorescent protein undergoes a 5-fold decrease in cyan fluorescence and a 300-fold increase in green fluorescence, resulting in an optical contrast of 1,500-fold. Because the resolution that can be obtained using photoactivated tags is largely dependent on the optical contrast triggered by photoactivation, PS-CFP should allow investigators to resolve finer changes in protein trafficking without complications caused by self-association of the tag.

Another advantage of PS-CFP over photoactivatable proteins such as PA-GFP is that it possesses significant fluorescence before photoactivation. This can be particularly helpful in cases where the protein is localized to discrete cellular regions. Lukyanov and colleagues took advantage of PS-CFP's preactivation fluorescence and stability at low pH to track the movement of tagged human dopamine transporter (hDAT) within filopodia (Fig. 1) and endosomes. They were also able to successfully photoactivate selected endosomes and track their movement in the cytoplasm. When two activated and nonactivated endosomes made contact, the authors observed, for the first time ever, the direct mutual exchange of cargo proteins between these cellular compartments, thus highlighting the value of PS-CFP for such studies.

Figure 1: A living endothelial cell transiently expressing the human dopamine transporter (hDAT) fused with the new photoswitchable cyan fluorescent protein (PS-CFP).
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The transporter is localized to the integral membrane, cellular processes and constitutive endosomes. A subpopulation of PS-CFP was selectively photoswitched at the cell edge.

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As a further aid in localization and tracking, PS-CFP can be visualized with standard ECFP and FITC filters, making it straightforward to use in multilabel experiments with red fluorophores. A potential problem with PS-CFP is that unlike Kaede, which uses a different wavelength for photoactivation and visualization of the nonactivated form, PS-CFP uses the same wavelength at different intensities for both processes. PS-CFP is also slightly more sensitive to bleaching than EGFP, which could further complicate some experiments. However, the authors clearly demonstrate that as long as researchers are careful in their experiments, these drawbacks do not obviate the significant advantages afforded by this new addition to the photoactivatable fluorescent protein family.