ABSTRACT
For understanding the rules and laws of adaptive immunity, high-throughput profiling of T-cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T-cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V-gene and J-gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.
Subject(s)
Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Immunity, Cellular/physiology , T-Lymphocyte Subsets/immunology , Animals , Mice , T-Lymphocyte Subsets/cytologyABSTRACT
Fluorescent proteins with emission wavelengths in the near-infrared and infrared range are in high demand for whole-body imaging techniques. Here we report near-infrared dimeric fluorescent proteins eqFP650 and eqFP670. To our knowledge, eqFP650 is the brightest fluorescent protein with emission maximum above 635 nm, and eqFP670 displays the most red-shifted emission maximum and high photostability.
Subject(s)
Biotechnology/methods , Luminescent Proteins , Whole Body Imaging/methods , Amino Acid Sequence , Animals , Biotechnology/instrumentation , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Escherichia coli/genetics , Escherichia coli/growth & development , HeLa Cells , Humans , Infrared Rays , Luminescent Proteins/genetics , Luminescent Proteins/toxicity , Mice , Molecular Sequence Data , Protein Multimerization , Protein Stability , Sequence Alignment , Transfection , Xenopus laevis/genetics , Xenopus laevis/metabolism , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
BACKGROUND: In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings. RESULTS: Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter). CONCLUSION: The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Green Fluorescent Proteins/chemistry , Luminescent Proteins/chemistry , Apoptosis , Caspase 3/metabolism , Escherichia coli/metabolism , Microscopy, Fluorescence/methods , Spectrometry, Fluorescence/methods , Red Fluorescent ProteinABSTRACT
The wild type red fluorescent protein eqFP578 (from sea anemone Entacmaea quadricolor, λ(ex) = 552 nm, λ(em) = 578 nm) and its bright far-red fluorescent variant Katushka (λ(ex) = 588 nm, λ(em) = 635 nm) are characterized by the pronounced pH dependence of their fluorescence. The crystal structures of eqFP578f (eqFP578 with two point mutations improving the protein folding) and Katushka have been determined at the resolution ranging from 1.15 to 1.85 Å at two pH values, corresponding to low and high level of fluorescence. The observed extinguishing of fluorescence upon reducing pH in eqFP578f and Katushka has been shown to be accompanied by the opposite trans-cis and cis-trans chromophore isomerization, respectively. Asn143, Ser158, His197 and Ser143, Leu174, and Arg197 have been shown to stabilize the respective trans and cis fluorescent states of the chromophores in eqFP578f and Katushka at higher pH. The cis state has been suggested as being primarily responsible for the observed far-red shift of the emission maximum of Katushka relative to that of eqFP578f.