 |
Fluorescent Proteins in Nature and as in vivo Imaging Tools
Summary: The major fields of Sergey Lukyanov's scientific interest center on molecular genomics methods, such as gene discovery and analysis, gene expression analysis, cDNA subtractive hybridization, and SNP detection; another focus is on identification of GFP-like fluorescent proteins and their development and applications.
One of our main interests is the search for and study of fluorescent proteins (FPs), particularly homologues of the Aequorea victoria green FP (GFP) and the development of enhanced versions for live object imaging. FPs are widely used as noninvasive probes to study various biological models, from individual cells to whole organisms. FPs enable one to track every step in the functioning of the protein of interest: expression, localization, movement, interaction, and activity in the cell, tissue, or organism. FP-based fluorescent sensors are applied mainly to visualize target gene promoter up- and down-regulation, label protein, detect protein-protein interactions, track protein movement, and monitor cellular parameters.
In 1999, we first cloned GFP-like FPs from Anthozoa species, including the well-known DsRed and other proteins; they are characterized by various spectral properties ranging from cyan to red fluorescence. Since then, we have been working intensely in the field, and today we are one of the leading groups studying the biochemistry of FPs and seeking to develop novel FPs with enhanced characteristics. Discovery of fluorescent and nonfluorescent GFP-like proteins in nonbioluminescent coral polyps disproved a commonly held notion that such proteins function only as components of bioluminescent systems. This discovery revealed the nature of fluorescent and nonfluorescent coral coloration, which until then could not be explained.
Later, we cloned dozens of different GFP-like FPs and nonfluorescent chromoproteins form Anthozoa and Hydrozoa species. More recently, we reported GFPs from evolutionarily distant organisms, such as marine Pontellidae species (Arthropoda, Crustacea, Maxillopoda, Copepoda, and Pontellidae). Thus, the phylogenetic distribution of the GFP family turned out to be quite unusual, given that, from an evolutionary standpoint, Cnidaria and Arthropoda are very distant groups. Excluding direct horizontal gene transfer from jellies or corals to copepods, it can be concluded that GFP-like proteins evolved before the separation of Bilateria and Cnidaria; thus, almost any animal taxon can potentially contain GFP homologues, suggesting that GFP-like proteins are likely to occur in various animals.
A wide range of fluorescent mutant variants were generated by means of site-directed and random mutagenesis, resulting in a series of FPs that are widely used today all over the world. Besides routine FPs, we developed a number of sophisticated FP variants, as described below, which are used widely for cell biology and medical studies.
A fluorescent “timer” was developed, which changes color from blue to green and then to red with time. Development of proteins that change fluorescent color in the course of maturation allows one to obtain, from the fluorescence color and in retrospect, information about the time of promoter activation.
In recent years, great efforts were made to find or create FPs as far-red-shifted as possible, both to expand the palette of FPs and to reach a spectral window favorable for tissue light penetration (650 to 1,100 nm); shorter wavelengths are absorbed by blood hemoglobin and longer ones are absorbed by water, and light-scattering intensity drops off with increasing wavelength. We developed several far-red FPs, expanding the range of FPs and opening novel possibilities for monitoring normal and pathological processes on the level of the whole organism.
We developed a range of so-called photoactivatable FPs. These proteins are capable of a manyfold increase in fluorescence intensity at certain excitation-emission wavelengths in response to irradiation at a specific wavelength. This property can be used to “switch on” a fluorescent signal by a beam of focused light and then to track movement of labeled objects, such as cells, organelles, and individual proteins. Until recently, photobleaching techniques, such as fluorescence recovery after photobleaching and fluorescence loss in photobleaching, were the major tools for studying protein mobility. Photoactivatable FPs provide a much more precise, direct, and less damaging way to study proteins' movement “topography.” Such FPs also provide a wide range of potential photoactivatable protein applications in advanced microscopy, such as enhanced resolution and modulated fluorescence resonance energy transfer.
We are putting much effort into developing genetically encoded fluorescent sensors to detect pH and charged species (such as Ca2+, Cl-, membrane potential, specific proteins, and so forth) or to measure the activity of specific enzymes. In contrast to exogenous chemical probes, such sensors can be expressed within a stable cell line or transgenic animal, targeted at a specific organelle in a cell or expressed within a specific tissue in an animal, thus essentially expanding possibilities for cell, developmental, and physiological studies as well as for high-throughput screenings. Importantly, such sensors eventually can be developed for any particular molecular species or event, and our laboratory develops novel genetically encoded fluorescent biosensors based on FPs.
Recently, we developed the first genetically encoded photosensitizer, an FP capable of producing reactive oxygen species in response to light irradiation, which can be used for the chromophore-assisted laser inactivation of a protein of interest and for photodynamic therapy.
Our work on FP development has given impulse to novel FP studies and applications and dramatically expanded the scope of in vivo imaging and precise photolabeling. Thus, it has become possible to label and visualize up to four or five living objects simultaneously and monitor several cellular processes and their interconnection as well as to follow protein dynamics, interactions, and turnover rate and to measure various cellular parameters in vivo in cell cultures and transgenic animals.
Last updated August 2009
|
|
|
|
