title: An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.
abstract: We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradiation with UV or violet light (350-400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest.
authors.affiliation: Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.
authors.forename: Ryoko
authors.initials: R
authors.lastname: Ando
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hama
authors.forename: Miki
authors.initials: M
authors.lastname: Yamamoto-Hino
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mizuno
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology / Q000196:embryology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D016011:Normal Distribution; D051381:Rats; D017208:Rats, Wistar; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins