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.

Mitotic cell rounding and epithelial thinning regulate lumen growth and shape

“…To label cellular and subcellular structures, mRNAs encoding the following fusion proteins were injected at one-cell stage after being synthetized with the SP6 mMessenger mMachine kit (Ambion): H2B-mCherry or H2B-GFP (100-150 pg) 47 , Pard3-GFP or Pard3-RFP (50-75 pg) 48,49 , Lyn-EGFP (membrane-GFP 100-150 pg) 50 , membrane-mCherry (100-150 pg) 51 , Lifeact-RFP or Lifeact-GFP (150 pg) 52 , hZO1-GFP (100 pg) 53 , GFP and Kaede (50 pg) 54 . To interfere with endogenous expression, embryos were injected at one-cell stage with 1.6 ng per embryo of emi1 translation blocking morpholino (5 0 -GTAGTTTGGACACTTCATATTGAGG-3 0 ) (ref.…”

Morphometric Analysis of Huntington’s Disease Neurodegeneration in Drosophila

“…In our experience, the effect of introducing a second UAS transgene on the expression of another is generally minimal. For example, in one case, we expressed homozygous or heterozygous Kaede (a fl uorescent protein, [ 35 ] ) with one or two copies of the elav -GAL4 driver and compared the Kaede intensities in eye discs (Fig. 5a, b ).…”

Imaging Translation in Single Cells Using Fluorescent Microscopy

“…To investigate the role of local b-actin mRNA translation in growth cone turning, Holt and colleagues constructed an axonal translation reporter by fusing the photoconvertible protein, Kaede, to the b-actin 3 0 UTR, which contains cis-acting elements responsible for RNA localization and translational control (Leung et al 2006). Kaede is a fluorescent protein that was isolated from the stony coral, Trachyphyllia geoffroyi, and undergoes an irreversible photoconversion (green to red) on irradiation with UV light that results in a 2000-fold increase in red-to-green fluorescent signal (Ando et al 2002). In growth cones expressing the Kaede reporter, new translation was observed on the side of the growth cone that was closest to an external gradient of the netrin-1 guidance cue (Fig.…”