Sergey Lukyanov

Fluorescent proteins from nonbioluminescent Anthozoa species.

We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same β-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H2O2) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H2O2-sensing protein, OxyR. Using HyPer we monitored H2O2 production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H2O2 occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (ΔΨ) and a change in cell shape. We also observed local bursts in mitochondrial H2O2 production during ΔΨ oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H2O2 increase upon physiological stimulation. Using HyPer we detected temporal increase in H2O2 in the cytoplasm of PC-12 cells stimulated with nerve growth factor.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light

Green fluorescent protein (GFP) and GFP-like proteins represent invaluable genetically encoded fluorescent probes. In the last few years a new class of photoactivatable fluorescent proteins (PAFPs) capable of pronounced light-induced spectral changes have been developed. Except for tetrameric KFP1 (ref. 4), all known PAFPs, including PA-GFP, Kaede, EosFP, PS-CFP, Dronpa, PA-mRFP1 and KikGR require light in the UV-violet spectral region for activation through one-photon excitation—such light can be phototoxic to some biological systems. Here, we report a monomeric PAFP, Dendra, derived from octocoral Dendronephthya sp. and capable of 1,000- to 4,500-fold photoconversion from green to red fluorescent states in response to either visible blue or UV-violet light. Dendra represents the first PAFP, which is simultaneously monomeric, efficiently matures at 37 °C, demonstrates high photostability of the activated state, and can be photoactivated by a common, marginally phototoxic, 488-nm laser line. We demonstrate the suitability of Dendra for protein labeling and tracking to quantitatively study dynamics of fibrillarin and vimentin in mammalian cells.

Bright far-red fluorescent protein for whole-body imaging.

For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.

Bright monomeric red fluorescent protein with an extended fluorescence lifetime

Fluorescent proteins have become extremely popular tools for in vivo imaging and especially for the study of localization, motility and interaction of proteins in living cells. Here we report TagRFP, a monomeric red fluorescent protein, which is characterized by high brightness, complete chromophore maturation, prolonged fluorescence lifetime and high pH-stability. These properties make TagRFP an excellent tag for protein localization studies and fluorescence resonance energy transfer (FRET) applications.

[20] Suppression subtractive hybridization: A versatile method for identifying differentially expressed genes

Abstract A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.

A ubiquitous family of putative gap junction molecules.

References 1. Artavanis-Tsakonas S, Rand MD, Lake RJ: Notch signaling: cell fate control and signal integration in development. Science 1999, 284:770-776. 2. Artavanis-Tsakonas S, Matsuno K, Fortini ME: Notch signaling. Science 1995, 268:225-232. 3. Petcherski AG, Kimble J: LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway. Nature 2000, 405:364-368. 4. Lehmann R, Jimenez F, Dietrich U, Campos-Ortega J: On the phenotype and development of mutants of early neurogenesis in Drosophila melanogaster. Wilhelm Roux’s Archiv Dev Biol 1983, 192:62-74. 5. Xu T, Rebay I, Fleming RJ, Scottgale TN, Artavanis-Tsakonas S: The Notch locus and the genetic circuitry involved in early Drosophila neurogenesis. Genes Dev 1990, 4:464-475. 6. Kopan R, Nye JS, Weintraub H: The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development 1994, 120:2385-2396. 7. Smoller D, Friedel C, Schmid A, Bettler D, Lam L, Yedvobnick B: The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers. Genes Dev 1990, 4:1688-1700. 8. Bettler D, Pearson S, Yedvobnick B: The nuclear protein encoded by the Drosophila neurogenic gene mastermind is widely expressed and associates with specific chromosomal regions. Genetics 1996, 143:859-875. 9. Schuldt AJ, Brand AH: Mastermind acts downstream of Notch to specify neuronal cell fates in the Drosophila central nervous system. Dev Biol 1999, 205:287-295. 10. Mitchell PJ, Tjian R: Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 1989, 245:371-378. 11. Kodoyianni V, Maine EM, Kimble J: Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans. Mol Biol Cell 1992, 3:1199-1213. 12. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, Israël A: Signaling downstream of activated mammalian Notch. Nature 1995, 377:355-358. 13. Kato H, Taniguchi Y, Kurooka H, Minoguchi S, Sakai T, Nomura-Okazaki S, Tamura K, Honjo T: Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives. Development 1997, 124:4133-4141.

Photoactivatable fluorescent proteins

The fluorescence characteristics of photoactivatable proteins can be controlled by irradiating them with light of a specific wavelength, intensity and duration. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner. Here, we discuss the properties of the available photoactivatable fluorescent proteins and their potential applications.

A genetically encoded photosensitizer.

Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, a homolog of green fluorescent protein (GFP). KillerRed generates ROS upon irradiation with green light. Whereas known photosensitizers must be added to living systems exogenously, KillerRed is fully genetically encoded. We demonstrate the utility of KillerRed for light-induced killing of Escherichia coli and eukaryotic cells and for inactivating fusions to β-galactosidase and phospholipase Cδ1 pleckstrin homology domain.