TECHNICAL NOTE |
Correspondence to: Gordon L. Hager, Laboratory of Receptor and Gene Expression, Bldg 41, Room B602, National Cancer Institute, Bethesda, MD 20892-5055..
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Summary |
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In this study we sought to develop a method for the co-localization of proteins in living cells utilizing the enhanced green fluorescent protein (EGFP) and a red-shifted EGFP variant, EYFP (enhanced yellow fluorescent protein). EYFP was expressed as an unsubstituted molecule while EGFP was fused to NF1 (EGFP-NF1), a transcription factor found exclusively in the nucleus. The Leica TCS SP laser scanning confocal microscope was used. This microscope allows the user to monitor the emitted light at defined wavelengths owing to the presence of a monochrometer in the emission light path. pEGFP-NF1 and pEYFP were co-expressed in the same cell and excited with the 476-nm and 488-nm argon laser lines. To separate the EYFP and EGFP fluorescence, EGFP-NF1 emission was recorded between 496 and 505 nm. These wavelengths are on the left shoulder of the EGFP emission peak and exclude most of the EYFP fluorescence. The EYFP emission was followed between 670 and 754 nm, utilizing the tail of EYFP emission that extends well beyond that for EGFP. Under these conditions we obtained excellent discrimination between EYFP fluorescence and EGFP-NF1 emission. These observations demonstrate that EYFP- and EGFP-substituted chimeras can be used for simultaneous detection in living cells. (J Histochem Cytochem 46:10731076, 1998)
Key Words: green fluorescent protein, confocal microscopy, protein localization
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Introduction |
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The green fluorescent protein (GFP) has become a useful tool for the study of intracellular localization of proteins (
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Here we describe a method to study the co-localization of proteins in living cells using the EGFP and EYFP chromophores. EYFP was expressed as an unsubstituted molecule in mouse mammary carcinoma cells, where it is localized throughout the cell. The EGFP chromophore was fused to NF1 (EGFP-NF1), a transcription factor expected to localize within the nucleus (
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Materials and Methods |
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Plasmids
pEGFP-NF1 was constructed as follows. pCTF-1 ( E. coli and purified by double CsCl banding.
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Cell Culture and Transfections
For this study, 1471.1 cells (C127-derived mouse mammary tumor line) (
Microscopy
Images were collected on a Leica TCS SP laser scanning confocal microscope fitted with spectrophotometers for emission band wavelength selection. Both EYFP and EGFP were excited with the 476-nm and 488-nm laser lines from an argon laser, with laser intensity set at 9% of available power. For visualization of EGFP, the emission window was set at 496505 nm (Figure 1). For visualization of EYFP, the emission window was set at 670754 nm (Figure 1), taking advantage of the long tail of EYFP emission beyond that of EGFP. Confocal image stacks were combined as xy projection images and printed on a Tektronix 480X dye-sublimation color printer.
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Results |
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To determine whether EGFP and EYFP could be spectrally separated within the same cell, a vector containing a nuclear localizing EGFP fusion protein (Figure 3) and the unfused EYFP protein (Figure 3) were transfected into 1471.1 cells. The EGFP protein was fused to NF1, a transcription factor known to localize exclusively to the nucleus (
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Discussion |
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The use of the green fluorescent protein has rapidly become a valuable tool for the study of protein localization in living cells because it eliminates the need for fixation of cells, which can be plagued by artifacts. Localization of any cloned protein can potentially be observed without the need for antibodies to that protein simply by fusing the protein of interest to GFP. With the recent advent of other GFP variants, such as EYFP, direct localization of two different proteins in the same cell is now possible. In this study we have demonstrated that the fluorescence of EGFP and EYFP can be separated, by setting the emission window to exclude either EGFP or EYFP fluorescence. The emission windows used were based on the data in Figure 1 (supplied by Clontech) and were chosen to maximize emission of the chromophore under observation while minimizing the emission from the second chromophore. Although these experiments were done using a confocal microscope equipped with a monochrometer in the emission path, it should be possible to use a standard confocal microscope and appropriate filter sets.
It should also be noted that the excitation lines available in this experiment were far from optimal for selective excitation of EYFP and EGFP. In fact, a 513-nm line can be obtained from the argon laser with appropriate modification. This line corresponds precisely to the excitation maximum for EYFP (Figure 1). Under these conditions, simultaneous visualization of EYFP and EGFP should be even more effective.
Using this technique described here, it will now be possible to directly study the co-localization of two different proteins of interest within the same living cell. Finally, the recent report of a BFP variant that is 100-fold brighter than the original Y66H BFP (
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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We thank Charles Hemphill (Leica; Exton, PA) for providing the laser scanning confocal instrument with emission spectrophotometer used in these experiments. We acknowledge Clonetech (Palo Alto, CA) for permission to reproduce the EGFP and EYFP excitationemission spectra.
Received for publication April 8, 1998; accepted May 7, 1998.
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Literature Cited |
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