- Doing well in the shadow of GFP
Novel chemical labeling system offers alternative for live-cell imaging.
Live-cell imaging has benefited tremendously from the development of genetically encoded fluorescent probes. The most common of these are GFP and other proteins. However, the past 15 years also have seen the development of a variety of chemical labeling systems for live-cell studies.
Generally, these labeling systems fall within two broadly defined groups, based on fluorophores that either bind peptide motifs directly or that are conjugated to small molecules that bind to acceptor peptides. With the latter, the binding process typically does not alter the behavior of the fluorophores, whereas with the former, the behavior can be modulated, though this potential for imaging studies has gone largely untapped.
In the February issue of Nature Biotechnology, researchers with Carnegie Mellon University in Pittsburgh reported a new class of protein-dye reporters that exploit this ability to modify fluorophore behavior. These reporters generate fluorescence through interactions with otherwise dark molecules known as fluorogens. The fluorogen-activating proteins offer distinct advantages over fluorescent proteins: They are directly accessible during experiments, and investigators can modulate their chemistry to produce various reporting and sensing capabilities. Thus, they offer unique possibilities as a biosensing platform.
Researchers have described novel protein-dye reporters that generate fluorescence based on interactions with otherwise dark molecules known as fluorogens. Referred to as fluorogen-activating proteins, or FAPS, these reporters hold promise as a biosensing platform. The fluorescence microscopy images shown here demonstrate another advantage of the technique: using a single laser to enable multicolor imaging of cells expressing various FAPS. Cells expressing HL1.0.1-T01 or L5-MG were mixed and excitedat 488 nm. A differential interference contrast image and a merge are included forcomparison. Reprinted with permission from Nature Biotechnology.
Researcher Christopher Szent-Gyorgyi explained what makes this possible. “The cornerstone is an appreciation of the fluorescence enhancement that is possible when free rotation around the chromophore of certain fluorogens (molecular rotors) is physically constrained by binding to a protein,” he said. “Direct screening of a very large yeast surface-displayed single-chain antibodies library for enhanced fluorescence by fluorescence-activated cell sorting enabled us to isolate such fluorescent complexes.”
The researchers screened the library using thiazole orange and malachite green derivatives. Both thiazole orange and malachite green have been identified as fluorogens; studies have reported fluorescence activation when the former intercalates into DNA, or when the latter binds to a specific RNA aptamer. They screened using a flow cytometer and software from BD Biosciences of San Jose, Calif., ultimately identifying eight different fluorogen-activating proteins.
The process by which they achieved this alternated between slow and tedious and relatively quick and painless. They isolated the first fluorogen-activating protein quickly in the summer of 2004, but more than two years elapsed — bringing additional funding and resources — before they isolated the second.
Further investigation uncovered a number of advantages to using the technique. For example, using a variety of derivatives of malachite green, the researchers found that different combinations of fluorogens and fluorogen-activating proteins exhibited different binding affinities, fluorescence intensities and excitation/emission spectra. This means that a single fluorogen-activating protein reporter can provide various spectral readouts simply by using different fluorogens, a property well suited for pulse-chase experiments.
Another advantage is that multicolor imaging of cells expressing various fluorogen-activating proteins is possible using a single excitation laser — in large part because the secondary excitation peak of malachite green and many of its derivatives is sensitive to modulation by the fluorogen-activating protein.
Taking advantage of this sensitivity, the researchers demonstrated nonoverlapping green and red fluorescence reporting using a laser-scanning microscope from Carl Zeiss that was outfitted with a 63× objective, with excitation provided by a single 488-nm argon laser.
According to Szent-Gyorgyi, the technique allows them to add a nonfluorescent dye directly to living cells and — without further manipulation — label a genetically tagged protein extremely specifically at a brightness comparable to that of fluorescent proteins. Furthermore, they demonstrated that fluorescence color can be modulated in two general ways: by chemically modifying the fluorogen or through genetic variation of the single-chain antibodies.
The technique could benefit a range of applications. The researchers noted specifically that the fluorogen-activating proteins can be used as reagents for live-cell imaging of proteins expressed on the outer leaflet of the plasma membrane and within the secretory lumen, where other methods have fallen short. Expression of fluorescent proteins is relatively weak on the outer leaflet, a problem that is exacerbated by background signal resulting from ectopic expression within the cell.
Several chemical labeling systems can label cell surface proteins, Szent-Gyorgyi said, “but these are not homogeneous assays as is our method.” He added that neither fluorescent proteins nor chemical labels can readily distinguish among proteins expressed on the cell surface and within the secretory pathway; such distinctions are possible with the currently described technique by using membrane-impermeant and -permeant fluorogens, respectively.
The investigators are working to improve the function of the fluorogen-activating proteins within intracellular reducing environments, such as the cytosol and nucleus, to enable visualization of tagged proteins almost anywhere within the cell. This should be possible using directed evolution methods, Szent-Gyorgyi said. The group also is planning to synthesize/isolate new dye and protein combinations to provide new fluorescent colors and binding specificities for multicolor labeling and pulse-chase applications.
MORE FROM PHOTONICS MEDIA