Optical highlighter fluorescent proteins, such as photoactivatable GFP (PA-GFP), the green-to-red photoconverter Kaede, and the photoswitchable Dronpa, allow direct and controlled activation of distinct molecular pools of the fluorescent proteins within the cell. The investigator selects the population of photoactivated or photoconverted molecules to be followed, so dynamic behavior can be monitored over time, independent of other newly synthesized proteins. An ideal optical highlighter protein should be readily photoconvertable (through the process of fluorescence activation and/or emission wavelength shifts) to produce a high level of contrast, as well as being monomeric for optimum expression in the target system. Listed in this section are key references to many of the original articles describing the discovery and properties of optical highlighters.
A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297: 1873-7 (2002). The original article describing the T203H mutation that converts wild-type GFP into a photoactivatable protein that can be controlled by illumination in the ultraviolet or violet range. PA-GFP displays a dynamic range of approximately 100 with a much lower background than the parent.
Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S. and Lukyanov, K.A.
Kindling fluorescent proteins for precise in vivo photolabeling. Nature Biotechnology 21: 191-4 (2003). A report on mutagenesis of asCP, a chromoprotein from the sea anemone Anemonia sulcata, which resulted in the first photoswitchable protein that can be toggled on and off with illumination. The Kindling fluorescent protein is photoactivated with green light and quenched with blue light.
Photoswitchable cyan fluorescent protein for protein tracking. Nature Biotechnology 22: 1435-1439 (2004). Research describing the mutagenesis of an Aequorea coerulescens protein to generate the first report of a cyan-to-green irreversible optical highlighter. Named PS-CFP, this fluorescent protein features a 1,500-fold increase in green fluorescence upon photoconversion.
An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proceedings of the National Academy of Sciences (USA) 99: 12651-6 (2002). Atsushi Miyawaki and co-workers describe a new green-to-red optical highlighter derived from a fluorescent protein isolated in the stony coral, Trachyphyllia geoffroyi. Named Kaede, this protein features a 2,000-fold increase in the ratio of red to green signal upon photoconversion.
Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306: 1370-3 (2004). Photoswitching between dark and bright states with a monomeric green fluorescent protein (Dronpa) derived from coral was first described in this paper. In addition to demonstrating optical highlighter behavior, Dronpa was used to report shuttling of a fusion chimera between the nucleus and cytoplasm.
Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6: 233-8 (2005). Report of a green-to-red optical highlighter, KikGR, a tetrameric coral protein with a chromophore and properties similar to Kaede. In live cells, however, KikGR is more efficiently photoconverted and is brighter than Kaede.
Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K.D. and Nienhaus, G.U.
EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proceedings of the National Academy of Sciences (USA) 101: 15905-10 (2004). Named after the Greek goddess of the dawn, Eos fluorescent protein is another green-to-red optical highlighter that has proved quite useful in dynamics and superresolution microscopy.
Nienhaus, G.U., Nienhaus, K., Holzle, A., Ivanchenko, S., Renzi, F., Oswald, F., Wolff, M., Schmitt, F., Rocker, C., Vallone, B., Weidemann, W., Heilker, R., Nar, H. and Wiedenmann, J.
Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications. Photochemistry and Photobiology 82: 351-8 (2006). Improving upon the properties of Eos, the authors introduce a tandem dimer construct and demonstrate its utility for tracking and dynamics.
Gurskaya, N.G., Verkhusha, V.V., Shcheglov, A.S., Staroverov, D.B., Chepurnykh, T.V., Fradkov, A.F., Lukyanov, S. and Lukyanov, K.A.
Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotechnology 24: 461-5 (2006). Expanding on earlier work, the authors generate a monomeric green-to-red optical highlighter through mutagenesis efforts. The new protein, Dendra, was demonstrated to be useful in live-cell imaging applications.
Subach, F.V., Patterson, G.H., Manley, S., Gillette, J.M., Lippincott-Schwartz, J. and Verkhusha, V.V.
Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nature Methods 6: 153-9 (2009). As a complement to PA-GFP, this report introduces three mCherry derivatives that exhibit photoactivation properties. The authors demonstrate the utility of PA-mCherry1 in superresolution experiments.