Interactions of fluorescent mercurial compounds and acidic fluorochromes with isolated living cells and nuclei

Summary Two fluorescent mercurials (fluorescein mercuric acetate and merbromin) and two acidic fluorochromes (brilliant sulfoflavine and primuline) were tested as supravital fluorochromes and compared with the fluorescent probe for hydrophobic groups, 8-anilino-1-naphthalene-sulfonic acid (ANS). Neither the mercurials nor the acidic fluorochromes appeared to penetrate intact cells, but all of the dyes fluorochromed damaged cells in a characteristic fashion. Expriments were then undertaken on nuclei isolated in 0.25 M sucrose. The fluorescent mercurials produced fluorescence of the nuclear envelope and nucleoli. More generalized fluorescence was induced if nuclei remained for prolonged periods in saline solutions balanced for intact cells or in nuclei exposed to 0.2 N hydrochloric acid. Acidic fluorochromes produced a more generalized distributional pattern of fluorescence. Primuline produced substantially more intense nuclear fluorescence than brilliant sulfoflavine at equimolar concentrations. Considered as a whole, these results indicate that an examination of the interaction of fluorescent dyes with unfixed cellular components could prove to be a useful tool in cell biology, particularly in the investigation of nuclear function.Supported in part by GRS-FR-5394 to the Albany Medical College.     

A theory of fluorescence polarization decay in membranes.

Decay of fluorescence polarization after an impulsive excitation is correlated with wobbling motion of fluorescent molecules in membranes. The motion is characterized by two parameters, a "wobbling diffusion constant" and a "degree of orientational constraint" both of which can be determined directly from experimentally obtained decay. Detailed discussion, including theoretically calculated time-courses of polarization decay, is given for several types of molecules embedded in lipid bilayers; these types cover a large part of fluorescent probes available at present. The theory is useful for the analysis of fluorescence polarization decay in any system where the orientation of fluorophore is restricted by the surrounding structure.     

The use of disulfonatonaphthalimide fluorescent dyes for the fluorescence polarization immunoassay of steroids

A series of fluorescent disulfonatonaphthalimide derivatives of testosterone and estriol have been synthesized and their fluorescent properties investigated. The fluorescence lifetimes of these derivatives were higher than that of the unreacted fluorescent dye while the quantum yields were of the same order. The compounds were therefore compared in terms of their utilizability in steroid fluorescence polarization immunoassays. The assay sensitivity and precision with each compound is discussed in terms of the position, type, and length of the chemical "bridge" linking the steroid to the fluorescent dye. It is proposed that these fluorescent labels are highly appropriate to this type of immunoassay.     

A genetic bistable switch utilizing nonlinear protein degradation

Background

Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida. However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes.

Results

In this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence.

Conclusions

To increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments.     

A histochemical study of the primary catecholamines in the hypothalamic neurons of the rat in relation to the ontogenetic and sexual differentiation

Summary The alterations in the content of the primary catecholamines in the hypothalamus have been studied with the histochemical technique of para-formaldehyde induced fluorescence.In the adult normal rats, independent of the sex, the fluorescence is located in the cell bodies of a few arcuate neurons, around the perikarya of the arcuate, para-ventricular and supra-optic neurons, and in the nerve endings of the arcuate neurons in the median eminence.The appearance of the primary catecholamines takes place at the 20th day of gestation in the para-ventricular and arcuate-peri-ventricular regions. In the supra-optic nucleus the fluorescent nerve terminals are not seen before birth. In the outer layer of the median eminence the fluorescence develops around the 5th post-natal day. No sexual differences were observed in the maturation of the primary catecholamines during the ontogenic development of the rat.More fluorescent cell bodies and nerve endings are seen in the arcuate neurons during the late diestrus than during estrus. The number and intensity of the catecholamine fluorescent neurons in the arcuate nucleus increases during the pregnancy. Castration increases slightly the number and intensity of the fluorescent cell bodies in the arcuate nucleus, but it diminishes the fluorescence in the median eminence. The changes were compensated by a treatment with testosterone propionate. Hypophysectomy alone has no effect on the fluorescence of the hypothalamic neurons.Supported by a grant from The Finnish Medical Society Duodecim.     

Regulation of red fluorescent light emission in a cryptic marine fish

Introduction

Animal colouration is a trade-off between being seen by intended, intra- or inter-specific receivers while not being seen by the unintended. Many fishes solve this problem by adaptive colouration. Here, we investigate whether this also holds for fluorescent pigments. In those aquatic environments in which the ambient light is dominated by bluish light, red fluorescence can generate high-contrast signals. The marine, cryptic fish Tripterygion delaisi inhabits such environments and has a bright red-fluorescent iris that can be rapidly up- and down-regulated. Here, we described the physiological and cellular mechanism of this phenomenon using a neurostimulation treatment with KCl and histology.

Results

KCl-treatment revealed that eye fluorescence regulation is achieved through dispersal and aggregation of black-pigmented melanosomes within melanophores. Histology showed that globular, fluorescent iridophores on the anterior side of the iris are grouped and each group is encased by finger-like extensions of a single posterior melanophore. Together they form a so-called chromatophore unit. By dispersal and aggregation of melanosomes into and out of the peripheral membranous extensions of the melanophore, the fluorescent iridophores are covered or revealed on the anterior (outside) of the iris.

Conclusion

T. delaisi possesses a well-developed mechanism to control the fluorescent emission from its eyes, which may be advantageous given its cryptic lifestyle. This is the first time chromatophore units are found to control fluorescent emission in marine teleost fishes. We expect other fluorescent fish species to use similar mechanisms in the iris or elsewhere in the body. In contrast to a previously described mechanism based on dendritic fluorescent chromatophores, chromatophore units control fluorescent emission through the cooperation between two chromatophore types: an emitting and an occluding type. The discovery of a second mechanism for fluorescence modulation strengthens our view that fluorescence is a relevant and adaptive component of fish colouration.     

Specificity of antinuclear autoantibodies in diseases with systemic destruction of connective tissue]

By using the fluorescent antibody technique several patterns of antinuclear antibodies (ANA) were detected in the systemic connective tissue diseases, as well as in some other diseases and in healthy donors, depending on the character of nuclear fluorescence. Homogeneous nuclear fluorescence, homogeneous fluorescence free of nucleaolar fluorescence, ring-shaped fluorescence, lumpy fluorescence, selective nuclealar fluorescence, nuclear fluorescence in the form of long fine plexiform bands associated with fluorescence in the nuclear membrane region. The latter ANA pattern was found only in several forms of lupus erythematosus, and this fluorescence was different from the previously reported "reticular" and "filamentous" patterns.     

Lipid-phenolic radical adducts as a plausible mechanism of "plant ageing" pigment formation

Co-oxidation of chlorogenic acid, caffeic acid, aesculetin and lucigenin with linoleic acid and egg phosphatidyl choline leads to the formation of fluorescent polymer materials. The fluorescent products are more lipophylic, they have lower elution volumes on Sephadex LH-20 column than related phenols and they differ by their fluorescence and chromatographic properties considerably from polymer lipid peroxidation products. From the presence in the excitation fluorescence spectra of a band corresponding to the phenols it was concluded that the fluorophoric groups were similar in both cases. The data are discussed in terms of liquid phase peroxidation and the appearance of the fluorescent species are attributed to the production of molecular adducts as a result of lipid and phenoxyl radical recombination. The characteristics of products obtained are compared with properties of fluorescent "plant ageing" pigments accumulated in aged and damaged plant cells.     

A Super-Ecliptic,pHluorin-mKate2, Tandem Fluorescent Protein-Tagged Human LC3 for the Monitoring of Mammalian Autophagy

Tandem fluorescent protein-tagged LC3s that were comprised of a protein tag that emits green fluorescence (e.g., EGFP or mWasabi) fused with another tag that emits red fluorescence (e.g. mCherry or TagRFP) were used for monitoring the maturation step of mammalian autophagosomes. A critical point for this tandem fluorescent-tagged LC3 was the sensitivity of green fluorescence at an acidic pH. EGFP and mWasabi continue to emit a weak, but significant, fluorescence at a pH of approximately 6. To overcome this issue, we focused on super-ecliptic pHluorin, which is a more pH-sensitive GFP variation. The green fluorescence of EGFP and mWasabi in the cells was still observed at weakly acidic levels (pH 6.0–6.5). In contrast, the fluorescence of pHluorin was more significantly quenched at pH 6.5, and was almost completely abolished at pH 5.5–6.0, indicating that pHluorin is more suitable for use in a tandem fluorescent protein-tag for monitoring autophagy. A pHluorin-mKate2 tandem fluorescence protein showed pH-sensitive green fluorescence and pH-resistant far-red fluorescence. We therefore generated expression plasmids for pHluorin-mKate2-tagged human LC3 (PK-hLC3), which could be used as a modifier for LC3-lipidation. The green and far-red fluorescent puncta of PK-hLC3 were increased under starvation conditions. Puncta that were green-negative, but far-red positive, were increased when autolysosomes accumulated, but few puncta of the mutant PK-hLC3ΔG that lacked the carboxyl terminal Gly essential for autophagy were observed in the cells under the same conditions. These results indicated that the PK-hLC3 were more appropriate for the pH-sensitive monitoring of the maturation step of autophagosomes.     

Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

Superresolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Superresolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photoconvertible fluorescent protein, respectively, resulting in the fixation-resistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and reactivated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.     

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease,and 1 phosphatase

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.     

A fluorescent modification of the Sakaguchi reaction on arginine

Summary The reactionproduct of arginine with 2,4-dichloro--naphthol proved to be fluorescent. This fluorescence could be used for the histochemical localization of arginine. The modification described resulted in a long lasting yellow-orange fluorescence of various arginine-containing tissues.Thanks are due to Mr. H. van Kooten for making the photograph.     

Fluorescent protein-based methods for on-plate screening of gene insertion

Background

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.

Methodology/Principal Findings

Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.

Conclusions/Significance

The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening.     

Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 1. Comparison of various 3'-O-ester adenine nucleotide derivatives

Fluorescent 3'-O-acyl-substituted adenine nucleotide (dimethylamino)naphthoyl and trinitrophenyl groups were studied for binding to the ADP/ATP carrier in mitochondria and submitochondrial particles. The changes in fluorescence intensity and emission maximum are for the most part similar to those observed in nonaqueous solvents. The (dimethylamino)naphthoyl derivatives from a largely quenched aqueous state have a shortwave shift up to 85 nm and increase up to 90-fold (1,5 derivative), whereas the little quenched naphthoyl derivatives show a fluorescence decrease and the weakly fluorescent trinitrophenyl derivative shows only a small increase on binding. All derivatives are good inhibitors (K1 = 1-10 microM) of nucleotide transport. The fluorescence titrations have an apparent K1/2 = 2-7 microM. The fluorescence of the 1,5-DAN nucleotide is fully suppressed by bongkrekate but only partially suppressed by carboxyatractylate. The fluorescence response is much stronger in submitochondrial particles than in mitochondria. Both facts suggest fluorescent binding to the "m" state of the carrier site at the inner face of the membrane. 1,5-DAN-AMP shows a slightly more efficient binding than DAN-ADP or DAN-ATP.     

Identification and In Vivo Characterization of NvFP-7R,a Developmentally Regulated Red Fluorescent Protein of Nematostella vectensis

Background

In recent years, the sea anemone Nematostella vectensis has emerged as a critical model organism for comparative genomics and developmental biology. Although Nematostella is a member of the anthozoan cnidarians (known for producing an abundance of diverse fluorescent proteins (FPs)), endogenous patterns of Nematostella fluorescence have not been described and putative FPs encoded by the genome have not been characterized.

Methodology/Principal Findings

We described the spatiotemporal expression of endogenous red fluorescence during Nematostella development. Spatially, there are two patterns of red fluorescence, both restricted to the oral endoderm in developing polyps. One pattern is found in long fluorescent domains associated with the eight mesenteries and the other is found in short fluorescent domains situated between tentacles. Temporally, the long domains appear simultaneously at the 12-tentacle stage. In contrast, the short domains arise progressively between the 12- and 16-tentacle stage. To determine the source of the red fluorescence, we used bioinformatic approaches to identify all possible putative Nematostella FPs and a Drosophila S2 cell culture assay to validate NvFP-7R, a novel red fluorescent protein. We report that both the mRNA expression pattern and spectral signature of purified NvFP-7R closely match that of the endogenous red fluorescence. Strikingly, the red fluorescent pattern of NvFP-7R exhibits asymmetric expression along the directive axis, indicating that the nvfp-7r locus senses the positional information of the body plan. At the tissue level, NvFP-7R exhibits an unexpected subcellular localization and a complex complementary expression pattern in apposed epithelia sheets comprising each endodermal mesentery.

Conclusions/Significance

These experiments not only identify NvFP-7R as a novel red fluorescent protein that could be employed as a research tool; they also uncover an unexpected spatio-temporal complexity of gene expression in an adult cnidarian. Perhaps most importantly, our results define Nematostella as a new model organism for understanding the biological function of fluorescent proteins in vivo.     

Hypothesis testing via integrated computer modeling and digital fluorescence microscopy

Computational modeling has the potential to add an entirely new approach to hypothesis testing in yeast cell biology. Here, we present a method for seamless integration of computational modeling with quantitative digital fluorescence microscopy. This integration is accomplished by developing computational models based on hypotheses for underlying cellular processes that may give rise to experimentally observed fluorescent protein localization patterns. Simulated fluorescence images are generated from the computational models of underlying cellular processes via a "model-convolution" process. These simulated images can then be directly compared to experimental fluorescence images in order to test the model. This method provides a framework for rigorous hypothesis testing in yeast cell biology via integrated mathematical modeling and digital fluorescence microscopy.     

Use of a fluorescence proble for hydrophobic groups, anilinonaphthalene sulphonic acid, in the supravital study of unusual slug oocyte nucleoli

Synopsis Oocytes of the slug,Limax flavus, display cytoplasmic and nucleolar fluorescence in the presence of 8-anilino-1-naphthalene sulphonic acid, a fluorescent probe for hydrophobic groups. When nuclei are isolated in a balanced salt solution, or in 0.25 m sucrose, chromosomes also become fluorescent, but chromosomes never display fluorescence in intact cells. In the complex multiform nucleolus characteristic of slug oocyte nuclei, the nucleolar cap displays conspicuously high levels of fluorescence and patterns of differential fluorescence are obvious. It would appear that this and similar reagents could be of significant value in the investigation of nucleoli in supravital and unfixed preparations of isolated nuclei.     

Microfluorimetric quantitation of catecholamine turnover in the sympathetic neurons of rat

Summary The quantitative aspects of the formaldehydeinduced fluorescence and the turnover of catecholamines in the sympathetic neuronal perikaryon of different sympathetic ganglia were studied after a blockade of the amine synthesis with -methyltyrosine. The concentration of catecholamines was determined by microfluorimetric quantitation method. The half-life of catecholamines in sympathetic neuronal perikarya was short and depended on the ganglion studied. The turnover rate of catecholamines in sympathetic neurons was highest in superior cervical and lowest in coeliac ganglion. Brightly fluorescent fibers were still seen five hours after the amine synthesis blockade, whereas almost all cell bodies had lost their fluorescence. Also small intensely fluorescent cells were still brightly fluorescent after the follow-up period. Microfluorimetrically determined turnover of catecholamines gave more detailed information about the turnover of catecholamines in sympathetic nervous system when compared to the biochemical methods used earlier.     

Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein

Several versions of split green fluorescent protein (GFP) fold and reconstitute fluorescence, as do many circular permutants, but little is known about the dependence of reconstitution on circular permutation. Explored here is the capacity of GFP to fold and reconstitute fluorescence from various truncated circular permutants, herein called "leave-one-outs" using a quantitative in vivo solubility assay and in vivo reconstitution of fluorescence. Twelve leave-one-out permutants are discussed, one for each of the 12 secondary structure elements. The results expand the outlook for the use of permuted split GFPs as specific and self-reporting gene encoded affinity reagents.     

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.