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Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2

Nature Protocols volume 2, pages 2024–2032 (2007)Cite this article

Abstract

A number of photoactivatable GFP-like fluorescent proteins (PAFPs) have been reported whose fluorescence can be switched on or whose fluorescent state can be modified by relatively intense irradiation at a specific wavelength. The use of these proteins gives unique opportunities to photolabel and track fusion proteins in a living cell. Here, we provide a protocol for the primary visualization, photoactivation and tracking of two monomeric PAFPs recently developed in our lab. Both these proteins, PS-CFP2 and Dendra2, are fluorescent and can be visualized before photoactivation. Upon photoactivation, their excitation and emission spectra undergo a dramatic red shift. The brightness of their initial and photoconverted states, along with the high dynamic ranges of both proteins, make them an attractive tool for protein photolabeling. Excluding genetic constructs cloning, cell culturing and transfection, the whole protocol may take anywhere from 10 min to several hours, depending on motility of the protein being studied.

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Figure 1: Photoconversion mechanisms for PS-CFP2 and Dendra2.
Figure 2: Spectral properties of PS-CFP2 and Dendra2, normalized spectra.
Figure 3: Photoactivation of PS-CFP2 within two HeLa cells.
Figure 4: Comparison of Dendra2 photoconversion by 488- and 405-nm lasers.
Figure 5: Photoactivation of Dendra2-vimentin with 488-nm laser.

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Acknowledgements

We are grateful to Elena Filinova for the help. This work was supported by Grants from Molecular and Cell Biology Program RAS, EC FP-6 Integrated Project LSHG-CT-2003-503259, Howard Hughes Medical Institute grant HHMI 55005618 and from the National Institutes of Health (GM070358). D.M.C. and K.A.L. are supported by Grants of the President of Russian Federation MK-8236.2006.4 and Russian Science Support Foundation.

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  1. Laboratory of Molecular Technologies for Biology and Medicine, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow, 117997, Russia

    Dmitriy M Chudakov, Sergey Lukyanov & Konstantin A Lukyanov

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  1. Dmitriy M Chudakov
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  3. Konstantin A Lukyanov
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Correspondence to Konstantin A Lukyanov.

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Authors have interest in Evrogen JSC, which commercializes PS-CFP2 and Dendra2.

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Chudakov, D., Lukyanov, S. & Lukyanov, K. Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat Protoc 2, 2024–2032 (2007). https://doi.org/10.1038/nprot.2007.291

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