Additional colors of fluorescent proteins enable better visualization in cellular and molecular biology.
Since 1994, when Douglas Prasher and Martin Chalfie demonstrated that the natural bioluminescence of the jellyfish A. victoria could be adapted to illuminate the sensory neurons in the unrelated species C. elegans, green fluorescent protein (GFP) and its genetic derivatives have been essential tools for cell biologists and other biomedical researchers. In 1999, another group of fluorescent proteins (FPs) was cloned from organisms inhabiting coral reefs and tide pools. The so-called reef FPs offer new photophysical properties, increasing the spectrum of color emissions available. Today, techniques based on FPs are among the most widely-used tools for studying cellular structure, function, and dynamics by focusing on the interaction among metabolites, proteins, and nucleic acids in biological systems.
FPs have two major functional roles in cellular and molecular biology. The first is as a reporter or marker, a probe genetically fused to a gene of interest, allowing scientists to determine whether it has been taken up by or expressed in the specimen. Such methodologies can be used to “light up” a selected protein, feature, or region of a cell, illuminating when certain proteins are formed, their locations, and where and when they move. This technique can also be used for quantitative assays and tracking. For instance, by using a protein that is localized to the nucleus, scientists are able to quantify a population of cells by counting the number of nuclei in a sample, and they can track the movement of individual cells that are labeled with the FP.
The second major function of FPs is serving as biosensors for the elucidation of such cellular processes as calcium wave induction, cyclic nucleotide messenger effects, membrane potential fluctuations, phosphorylation, and intracellular protease action. Indicators for cyclic AMP (adenosine monophosphate), pH, phosphorylation, and calcium, among others, are widely used in laboratories today. Of special interest is a class of proteins used in Förster (also sometimes called fluorescence) resonance energy transfer (FRET), a technique most commonly used to monitor ratiometric changes that indicate dynamic processes in living tissue.
FRETting with FPs
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FRET describes an energy transfer mechanism between a pair of chromophores that respond to different excitation wavelengths. First, a fluorescent donor is excited at its specific fluorescence wavelength. This excited energy is then transferred to a nearby acceptor molecule, and the donor returns to its ground state. For instance, among the most popular biosensors for calcium ion detection is Cameleon, which fuses a pair of proteins, one cyan and one yellow, to a linking peptide that is responsive to calcium ions. As the calcium ion concentration increases, the fluorescence of the cyan protein decreases and that of the yellow protein increases. The change in color is immediately visible and represents a sensitive ratiometric indicator for the concentration of calcium ions.
FRET works particularly well with HeLa and other immortalized cell lines, partly because they are transfected with high efficiency; that is, it is relatively easy to insert a bit of genetic material (the FP) into them. Transfection is often accomplished by mixing a lipid-based reagent with the FP DNA to produce liposome-like structures, which then fuse with the target cell membrane and deposit the DNA inside. But in fact, effective transfection remains one of the greatest hurdles facing scientists working with FPs. Many specimens, especially the important primary and stem cells, still present major issues ranging from low transfection rates to large-scale cell mortality.
Researchers are now experimenting with using benign viruses to transport the genetic material into cells more effectively. Baculoviruses, a family of large rod-shaped viruses that typically infect insects, but do not replicate in vertebrate animal cells, are among the most promising. In addition to having the advantage of not harming the mammalian cells that are common subjects of study, they also are considered safe for laboratory personnel to handle.
Recombinant baculoviruses in which the polyhedrin promoter has been replaced with a mammalian promoter, termed BacMam viruses, were originally designed as potential new gene therapy delivery vehicles. Invitrogen, Carlsbad, Calif., recently introduced a line of FP-based biosensors that fuse BacMam virus genomes with Cameleon DNA for intracellular calcium signal measurements. Based on the version of the GFP-derived sensor family developed by Roger Tsien at the Univ. of California, San Diego, the Premo Cameleon sensor features cyan and yellow FPs in close proximity, allowing FRET to occur.
In addition to viral delivery mechanisms, there have been substantial gains over the past few years in the development of new FPs that cover a much larger portion of the visible light spectrum. Research efforts also have led to improvements in the brightness and photostability of FPs, enhancing their utility for research. With better understanding of the peptide-based fluorescent chromophores and their interaction with the backbone polypeptide structures, it gradually becomes simpler to fine-tune high-performance color variants and further broaden the range of colors available.
All colors of the rainbow
GFP and its more-widely used variant, enhanced GFP (EGFP), are already well known, but a variety of additional proteins emitting in the green portion of the spectrum have been isolated. Among these is AceGFP, from Evrogen, Moscow, Russia, a bright green protein that may have potential FRET application when used with one of the new red FPs, though its advantages (if any) over EGFP are not fully understood at this time. Other new green FP variants are commercially available, including Azami Green, (MBL International, Woburn, Mass.) and Emerald (Invitrogen).
Yellow FPs (YFPs) include the enhanced variant (EYFP), which is one of the brightest proteins. Though most existing YFPs demonstrate sensitivity to low pH and chloride ions, compromising their utility in acidic organelles (such as the Golgi, peroxisomes, and lysosomes), some exciting new yellow derivatives are now available. Citrine expresses at much higher levels in mammalian cultures than many previous yellows and is much brighter, though less photostable, than EGFP. Venus is less acid-sensitive than EYFP and has been demonstrated in numerous applications, although rapid photobleaching compromises long-term imaging. Even brighter than Citrine or Venus is YPet, which was developed specifically for FRET applications together with the Cyan protein CyPet. Other promising Cyan FPs (CFPs) include Cerulean, AmCyan1 (Clontech, Mountain View, Calif.), MiCy (MBL International), and mTFP1 (Allele Biotechnology, San Diego, Calif).
Blue FPs (BFPs), long neglected due to poor photostability and limited brightness, have undergone a recent surge in interest. BFPs have broad emission peaks and have already been coupled into biosensors along with GFPs to monitor apoptosis, calcium flux, and other phenomena. However, BFPs must be excited by UV light, which is highly toxic to most cells and requires specialized microscope illumination sources and filters. In addition, this region of the spectrum often features significant scattering, rapid photobleaching, and high levels of autofluorescence. However, some of the most important recent developments are in the area of bringing new BFPs to market including Azurite, available from addgene.com, one of the brightest and most photostable BFPs yet developed.
A large variety of red and orange FPs (RFPs and OFPs, respectively) has been isolated from coral reef species. Some of the most significant oranges include Kusabira Orange (KO) and its derivative mKO. These are bright and stable, making mKO, in particular, useful for long-range imaging studies. Other key oranges include mOrange and tandem dimer Tomato, which can be linked with Sapphire as a FRET pair. RFPs are a desirable alternative because the use of longer wavelengths of light allows imaging deeper within specimens, causing less damage. However, RFPs have been difficult to develop and continue to present problems with incomplete maturation, lack of brightness, and reduced photostability compared to EGFP.
One key group of red proteins includes DsRed and its variants, DsRed2 and DsRed-Express, already being used as markers for gene expression and cell tracking in vivo. Other important reds including mCherry, mStrawberry, HcRed1 and the far-red eqFP611, have further expanded the horizon for RFPs.
Finally, there has been great interest in KillerRed, a new FP photosensitizer capable of generating reactive oxygen species upon light irradiation and which, upon prolonged illumination, will cause death in the host cell.
Optical highlighters
Of additional interest are photomarkable FPs, often referred to as optical highlighters, which work well in demonstrating protein turnover and dynamics. Among the most useful highlighters are photoactivatable GFP and PA-mRFP1, reversibly photoswitchable Dronpa, the tetrameric kindling FP (KFP), green-to-red photoconvertible proteins Kaede, KikGR, and EosFP, and cyan-to-green photoconvertible monomer PS-CFP.
One of the key limitations of FPs is their size—often larger than the host protein to which they are fused. GFP, for instance, is 238 amino acids in size. An alternative approach is to tag the host protein with a small peptide. Its core sequence is only 6 residues long and contains one pair of cysteines, proline, glycine, and another pair of cysteines that can be labeled in living cells with dyes capable of crossing the plasma membrane. This system, originally developed by Roger Tsien and his team, has already found many applications in cell biology and was recently used in conjunction with FPs to examine the behavior of the Golgi apparatus in dividing cells.
—Ilene Semiatin
Ilene Semiatin is a freelance writer based in White Plains, N.Y.
