Abstract
In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported1,2,3,4,5,6,7, including photoactivatable PA-GFP1, photoactivatable protein Kaede2, the DsRed 'greening' technique3 and kindling fluorescent proteins6,7. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.
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Acknowledgements
We are grateful to Alexander Sorkin for cDNA of hDAT and valuable discussions. We thank Natalia E. Yelina for the critical reading of the manuscript. This work was supported by grants from National Institutes of Health/National Institute on Drug Abuse A DA014204 and National Institutes of Health/National Institute of General Medical Sciences GM070358 (to V.V.V.), Russian Academy of Sciences for the program “Molecular and Cell Biology” and EC FP-6 Integrated Project LSHG-CT-2003-503259.
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Supplementary information
Supplementary Fig. 1
PS-CFP Ph stability before and after photoswitching compared to EGFP. (PDF 311 kb)
Supplementary Fig. 2
Photostability of the photactivated PS-CFP compared to EGFP. (PDF 322 kb)
Supplementary Fig. 3
Protein gel electrophoresis analysis of PS-CFP. (PDF 181 kb)
Supplementary Fig. 4
PS-CFP-actin fusion. (PDF 115 kb)
Supplementary Fig. 5
PS-CFP photoswtiching in the cell fusion. (PDF 171 kb)
Supplementary Table 1
Photoactivatable GFP-like protein properties (PDF 7 kb)
Supplementary Video 1
SP-CFP-hDAT fusion movement tracking within filopodia I (MOV 817 kb)
Supplementary Video 2
SP-CFP-hDAT fusion movement tracking within filopodia II (MOV 1020 kb)
Supplementary Video 3
SP-CFP-hDAT fusion movement tracking within early endosomes (MOV 723 kb)
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Chudakov, D., Verkhusha, V., Staroverov, D. et al. Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22, 1435–1439 (2004). https://doi.org/10.1038/nbt1025
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DOI: https://doi.org/10.1038/nbt1025
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