Measurement of intracellular magnesium concentration in 3T3 fibroblasts with the fluorescent indicator Mag-indo-1

Mag-indo-1, a fluorescent probe for measuring intracellular magnesium concentration, was used in 3T3 fibroblasts with microspectrofluorometry. The complex emission fluorescence spectrum emitted by a single living cell was analyzed with a computerized method, making it possible to evaluate the contribution of each species of Mag-indo-1 to the total fluorescence. The dye self-association observed in solutions at high dye concentration was not encountered in cells. The model of equilibria of Mag-indo-1 (monomer form) with protons, magnesium, and protein was then applied to calculate the intracellular magnesium concentration. The spectral analysis evaluated the contribution of each fluorescent species of Mag-indo-1 (a deprotonated, a magnesium-bound, and a protein-bound form) and of the cell autofluorescence to the total cell fluorescence. This method permitted accurate and reproducible measurements of intracellular magnesium concentration. Finally, this method was applied to the measurement of intracellular magnesium concentration in a 3T3 fibroblast population in exponential growth phase.     

Dual-function reporter protein for analysis of gene expression in living cells

The firefly luciferase (Luc) protein and the jellyfish green fluorescent protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Luc useful for a particular experimental application are quite distinct. A recombinant protein with both fluorescent and bioluminescent characteristics might take advantage of the strengths of both reporters. An expression vector encoding a chimeric protein in which GFP was tethered to Luc through a 19-amino acid linker was prepared and characterized. Western blotting with antibodies specific for either GFP or Luc showed that a protein of appropriate size was expressed in transfected cells. Fluorescence microscopy revealed bright green fluorescence from transfected cells, indicating proper formation of the GFP chromophore. Luc enzymatic activity in protein extracts from transfected cells showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the half-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantification of Luc activity from the same living cells.     

The application of double fluorescence to the rapid selection of atypical cervical epithelial cells spread in monolayers

Monolayer spreads of cervical cells were prepared and reacted in sequence with two fluorescent probes. The nuclei were reacted with 4,6-Diamidino-2-phenylindole (DAPI), resulting in white fluorescence of all cell nuclei. Those cells possessing active guanidinobenzoatase (GB) bound the second probe, rhodamine-alpha-N-agmatine (Rh-Agm), resulting in orange cell surface fluorescence. Atypical epithelial cells possessed both active GB and enlarged nuclei; such cells could easily be recognised by their cytological appearance. We illustrate our results in the form of colour prints which are representative of our observations of cells in both normal and abnormal cervical spreads.     

Aldol sensors for the rapid generation of tunable fluorescence by antibody catalysis

The synthesis of novel fluorogenic retro-aldol substrates for aldolase antibody 38C2 is described. These substrates are efficiently and specifically processed by antibody aldolases but not by natural cellular enzymes. Together, the fluorogenic substrates and antibody aldolases provide reporter gene systems that are compatible with living cells. The broad scope of the antibody aldolase allows for the processing of a range of substrates that can be designed to allow fluorescence monitoring at a variety of wavelengths. We also have developed the following concept in fluorescent protein tags. beta-Diketones bearing a fluorescent tag are bound covalently by the aldolase antibody and not other proteins. We anticipate that proteins fused with the antibody can be tagged specifically and covalently within living cells with fluorophores of virtually any color, thereby providing an alternative to green fluorescent protein fusions.     

The use of exogenous fluorescent probes for temperature measurements in single living cells

The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and 6-dodecanoyl-2-dimethylamino-naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat-induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane-bound NBD-conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature-dependent Stokes shift as the membrane undergoes a gel-to-liquid-crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady-state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2 degrees C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1 degree C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short-pulse microirradiation on single cells.     

Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.     

Detection of undegraded oligonucleotides in living sea urchin eggs by fluorescence resonance energy transfer

A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. A 10-meric oligodeoxynucleotide labeled with fluorescein at its 5'-end and with rhodamine X at its 3'-end (F-ODN-R) was used. The oligomer had a specific absorption spectrum with peaks at 497 nm and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, F-ODN-R had a specific fluorescence spectrum with peaks at 523 nm and 610 nm. The digestion of F-ODN-R with an endonuclease caused the increase in light intensity at 523 nm and the decrease at 610 nm. To examine effects in vivo, living sea urchin eggs were injected with a solution of F-ODN-R and excited with blue light at 470-490 nm. Two fluorescent images, a green image at 520-560 nm and a red image at above 580 nm, were obtained when a single egg was viewed under a fluorescence microscope. Eggs injected with the digested F-ODN-R emitted only green fluorescence. These results indicated that the integrity of oligonucleotides can be estimated in living cells by monitoring FRET after double-labeling of the oligonucleotides with fluorescein and rhodamine X.     

Comparison of activation versus induction of unresponsiveness of virus-specific CD4+ and CD8+ T cells upon acute versus persistent viral infection

The green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C-terminal endoplasmic reticulum (ER)-retention motif of mGFP5-ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18-24 h) depending on the cell type. Most chloroplast-rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast-poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non-acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.     

Translocation of phosducin in living neuroblastoma x glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein

Activation of G protein-coupled receptors triggers translocation of certain proteins from cytoplasm to cell membrane located targets. One of these cytosolic proteins is phosducin (Phd) which has been described to compete with G protein-coupled receptor kinases for Gbetagamma dimers attached to the cell membrane, thereby attenuating desensitization of activated receptors. These features of protein redistribution prompted us to examine whether stimulation of membrane associated E-prostaglandin receptors coupled to Gs causes Phd to migrate towards the plasma membrane. We made use of enhanced green fluorescence protein (EGFP), a reporter protein, to follow redistribution of Phd both by means of confocal microscopy and biochemical techniques in living neuronal NG 108-15 hybrid cells challenged with prostaglandin E1 (PGE1). The cells were transiently transfected to express Phd fused to the C-terminus of EGFP, or to express EGFP only. Overexpression of the proteins is implied by FACS analysis as well as by western blot technique, and the functional integrity of EGFP-tagged Phd was confirmed by its ability to elevate cAMP accumulation. Time-lapse imaging of single living cells by means of confocal microscopy revealed that exposure to prostaglandin causes EGFP/Phd, which is evenly spread throughout the cell, to relocate towards the membrane within few minutes. Fluorescence associated with the cell nucleus displayed little rearrangement. The principle finding that prostaglandin triggers translocation of Phd from cytosol to the cell periphery was verified with membranes prepared from EGFP/Phd expressing cells. We found maximal concentrations of membrane associated fluorescent material 5 to 7 min upon prostaglandin exposure. The present study reports for living NG 108-15 hybrid cells that PGE1 stimulation causes cytosolic Phd to translocate towards the membrane, where it is believed to bind to G protein subunits such as Gbetagamma and Galphas.     

Development and applications of enhanced green fluorescent protein mutants

The introduction of several mutations resulted in the generation of improved mutants of the green fluorescent protein (GFP). A strong green (GFPsg25) and blue (BFPsg50) fluorescent protein, gave 50-fold-100-fold brighter fluorescence compared to wild-type GFP and BFP (Tyr66His), respectively, upon expression in mammalian cells. GFPsg25 and BFPsg50 have different excitation and emission maxima. This allows their use as an efficient dual-color tagging system and their independent detection in living cells.     

Confocal laser scanning microscopy for the study of membrane transporter proteins

Confocal laser scanning microscopy, in combination with fluorescent probes including fluorescence labelled antibodies, enables us to obtain optical sectioning images of fixed and living biological samples. It is a powerful method for studies of membrane transporter proteins in that it provides information on the spatial distribution of not only the transporter proteins themselves, but also the molecules and ions they transport. In the near future, it may also provide information on molecular interaction between and conformation changes of various proteins with the aid of new type of fluorescent probes which utilize the fluorescence resonance energy transfer phenomenon.     

A flow cytometric method for rapid selection of novel industrial yeast hybrids

We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 x 10(6) cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains.     

Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein

The green fluorescent protein (GFP) from Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of cells and organisms. Despite many early successes using this reporter, wild type GFP is suboptimal for most applications due to low fluorescence intensity when excited by blue light (488 nm), a significant lag in the development of fluorescence after protein synthesis, complex photoisomerization of the GFP chromophore and poor expression in many higher eukaryotes. To improve upon these qualities, we have combined a mutant of GFP with a significantly larger extinction coefficient for excitation at 488 nm with a re-engineered GFP gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yield an enhanced GFP which provides greater sensitivity in most systems.     

Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells

Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.     

Fluorescamine, a fluorescence probe for amino groups in histochemical studies of plant cells and the effect of mercury fixation

When fixed in mercuric chloride solutions and stained with Fluorescamine, histological plant specimens emit a strong fluorescence. The fluorophore distribution is topologically identical to the staining pattern revealed by visible light methods for nucleoproteins, but the fluorescence mode of viewing preparations gave greater sensitivity and contrast than transmitted light absorption methods. The parameters that influence the formation of the fluorescent image in plant cells are discussed. The results obtained indicate that the mercury-Fluorescamine reaction is an ideal histochemical procedure for collecting qualitative and analytical information on plant nuclei and on the changes of nucleolar architecture that occur during the cellular developmental cycle.     

Real-time optical monitoring of ligand-mediated internalization of alpha1b-adrenoceptor with green fluorescent protein

The study of G protein-coupled receptor signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We show here that a fully functional alphalb-adrenoceptor tagged with the green fluorescent protein (alphalbAR/GFP) can be used to determine the molecular mechanism of intemalization of alphalbAR/ GFP in living cells. In mouse alphaT3 cells, alpha1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent receptor binds agonist and antagonist and stimulates phosphatidylinositol/Ca2+ signaling in a similar fashion to the wild receptor. In addition, alpha1bAR/ GFP can be internalized within minutes when exposed to agonist, and the subcellular redistribution of this receptor can be determined by measurement of endogenous fluorescence. The phospholipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca2+-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted alphalbAR/GFP redistribution. Agonist-promoted internalization of alphalbAR/GFP was closely linked to phospholipase C activation and was dependent on protein kinase C activation, but was independent of the increase in intracellular free Ca2+ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of alphalbAR (as well as other G protein-coupled receptors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced receptor endocytosis.     

A method for detection of hydroxyl radicals in the vicinity of biomolecules using radiation-induced fluorescence of coumarin

A novel method is described to quantitate radiation-induced hydroxyl radicals in the vicinity of biomolecules in aqueous solutions. Coumarin-3-carboxylic acid (CCA) is a non-fluorescent molecule that, upon interaction with radiation in aqueous solution, produces fluorescent products. CCA was derivatized to its succinimidyl ester (SECCA) and coupled to free primary amines of albumin, avidin, histone-H1, polylysine, and an oligonucleotide. When SECCA-biomolecule conjugates were irradiated, the relationship between induced fluorescence and dose was linear in the dose range examined (0.01-10 Gy). The fluorescence excitation spectrum of irradiated SECCA-biomolecule conjugates was very similar to that of 7-hydroxy-SECCA-biomolecule conjugates, indicating the conversion of SECCA to 7-hydroxy-SECCA following irradiation. Control studies in environments that excluded certain radiation-induced water radicals for both the conjugated and unconjugated forms of irradiated SECCA demonstrated that: (1) the induction of fluorescence is mediated by the hydroxyl radical; (2) the presence of oxygen enhances induced fluorescence by a factor of about 1.4, and (3) other primary water radicals and secondary radicals caused by interaction of primary water radicals with biomolecules do not significantly influence the induced fluorescence. The data indicate that the induction of fluorescence on SECCA-biomolecule conjugates records specifically the presence of the hydroxyl radical in the immediate vicinity of the irradiated biomolecule. The method is rapid and sensitive, uses standard instrumentation, and the sample remains available for further studies.     

Chemical study of the topography of porcine lutropin (LH) using dinitrofluorobenzene and dansyl chloride

Treatment of porcine lutropin beta-subunit by increasing amounts of dansyl chloride shows that only one fluorescent group can be bound to the free subunit, namely on tyrosine beta-37. This modification prevents reassociation with native chi-subunit. In contrast to dansyl chloride, dinitrofluorobenzene reacts preferentially with tyrosine beta-59. This substitution does not interfere with the reassociation with the native chi-subunit. By using equimolar ratio of dansyl chloride and porcine lutropin chi-subunit it is possible to modify this subunit on a single site, which is found to be a tyrosine residue. The monodansylated X-subunit is still able to recombine with native B-subunit but its fluorescence is found to be markedly quenched upon binding. In addition, the O-dansyl-tyrosyl fluorescence quenching by potassium iodide is more effective on the recombined dimer than on the free chi-subunit. Both reconstituted dimers (native xhi chi dinitrophenylated beta and dansylated chi chi native beta) are without biological activity. It is not clear whether the substituted phenolic groups are essential or whether the added groups prevent the chi beta dimer from taking the active conformation. This alternative is discussed in the light of recent data from this laboratory and others.     

A FLASH of insight into cellular chemistry: genetically encoded labels for protein visualization in vivo

Genetically encoded fluorescent labels, such as green fluorescent protein, make it possible to visualize a protein's natural distribution and environment in living cells. A new approach to protein labeling in living cells has been devised in which a small, membrane-permeable ligand binds with high affinity and specificity to a short peptide motif that can be incorporated into the protein of interest; the ligand becomes brightly fluorescent after binding to the peptide.     

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.