Nanosecond study of fluorescently labeled troponin C.

The time-resolved extrinsic fluorescence of rabbit skeletal troponin C was studied with the protein labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Both the intensity and anisotropy decays followed a biexponential decay law, regardless of the ionic condition, pH, viscosity or temperature. The lifetimes and their fractional amplitudes were insensitive to Mg2+, and the lifetimes were also insensitive to Ca2+. In response to Ca2+ binding to all four sites, the fractional amplitude (alpha 1) associated with the short lifetime (tau 1) decreased by a factor of two, thus increasing the ratio of the two amplitudes alpha 2/alpha 1 from 1.6 to 4.3. These amplitude changes suggest the existence of two conformational states of TnC-IAEDANS, with the conformation associated with the long-decay component (tau 2) being promoted by saturation of the two Ca(2+)-specific sites. At pH 5.2 the ratio alpha 2/alpha 1 for the apo-protein was 3.5 indicating different relative populations of the two decay components when compared with pH 7.2. In the presence of Ca2+ at the lower pH, alpha 2/alpha 1 decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca2+ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molecular motions that were not resolved in the present experiments, and some of these motions were sensitive to Ca2+ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of the central helix of troponin C.     

Diffusion of fluorescently labeled macromolecules in cultured muscle cells.

Myotubes were obtained from culture of satellite cells. They had a sarcomeric organization similar to that of muscle. The diffusion in the direction perpendicular to the fibers of microinjected fluorescein isothiocyanate-dextrans of molecular weight ranging from 9500 to 150,000 was examined by modulated fringe pattern photobleaching. On the time scale of the observation, 10-30 S, all of the dextrans were completely mobile in the cytoplasm. The diffusion coefficients were compared to the values obtained in water. The ratio D(cytoplasm)/D(w) decreased with the hydrodynamic radius R(h) of the macromolecules. The mobility of inert molecules in muscle cells is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments: D(cytoplasm)/D(w) = (D/D(w)) protein crowding x (D/D(w))(filament screening). The equation (D/D(w))filament screening = exp(-K(L)RCh) was used for the contribution of the filaments to the restriction of diffusion. A free protein concentration of 135 mg/ml, a solvent viscosity of cytoplasm near that of bulk water, and a calculated K(L) of 0.066 nm(-1), which takes into account the sarcomeric organization of filaments, accurately represent our data.     

Directed covalent immobilization of fluorescently labeled cytokines

Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokine--receptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a derivative of O(6)-alkylguanine-DNA alkyltransferase that couples itself covalently to benzylguanine. Bioactivities of the SNAP-YFP-cytokines were shown to be comparable with the nontagged cytokines. Efficient coupling of SNAP-YFP-cytokines to benzylguanine-modified beads was demonstrated by flow cytometry. The fact that enterokinase treatment released most of the fluorescence from the beads is indicative for directed coupling and only marginal adsorptive binding. Cellular responses to SNAP-YFP-cytokine beads were analyzed in cellular lysates and by confocal microscopy indicating that the directionally immobilized cytokines are fully signaling competent with respect to the activation of ERK and STAT3. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labeled proteins including the convenient and reliable control of coupling efficiency and orientation.     

Hydroxyl radical footprinting of fluorescently labeled DNA

Footprinting is one of the simplest and most accurate approaches to investigate structure and interaction of biopolymers. It is based on the more difficult accessibility of intra- and intermolecular contacts for external damaging agents. According to this method, one end of polymer molecules is labeled before a sample is incubated with a damaging agent. The distribution of split products is used to conclude on the accessibility of different polymer regions under specific conditions. A variety of enzymatic and chemical splitting agents are used for footprinting. Currently, the highest temporal and spatial resolution without profound specificity to a nucleotide sequence can be reached with the use of hydroxyl radicals. A new variant of this approach, which suggests the use of DNA fluorescent labeling together with the present-day quantitative analysis, will allow extending the method’s boundaries.     

Microsatellite genotyping of post-PCR fluorescently labeled markers

We show that a post-PCR multicolor fluorescence-labeling technique is applicable to multiplex microsatellite genotyping. Forty-three dinucleotide microsatellite markers, which are located on 11q13-23, a candidate region for dominant familial exudative vitreoretinopathy (FEVR), were used to evaluate the quality of the marker profile produced by this technique. Thirty-eight people from six families with this disease were subjects for genotyping. The samples revealed clearly separated fragment peaks with signal intensities sufficient for analysis. All genotypes were consistent with Mendelian inheritance within each family. This method is cost effective because it does not use expensive fluorescently labeled primers. It also has the advantage of avoiding ambiguity in the analysis, which may arise from the addition of non-template nucleotides by Taq DNA polymerase.     

Cellular penetration of fluorescently labeled superoxide dismutases of various origins.

BACKGROUND: Using fluorescently labeled superoxide dismutase (SOD) and flow cytometry, we have shown previously that the enzyme CuZn SOD (EC 1.15.1.1) from bovine erythrocytes binds rapidly to the cell surface with slow uptake into the cell during the following hours. The degree of labeling was most important for monocytes in comparison to other blood cells (erythrocytes, lymphocytes, and neutrophils) and fibroblasts. In agreement with the flow-cytometric findings, the inhibition of superoxide production was more important for SOD-pretreated monocytes than for neutrophils, as demonstrated with the cytochrome c reduction assay. It was thus of interest to confirm the observed differences between monocytes and neutrophils with confocal laser microscopy, study in greater detail the kinetics of binding, penetration, and intracellular localization of the enzyme, and compare the results obtained with bovine CuZn SOD with those from SODs of other origins and carrying different active sites. MATERIALS AND METHODS: Recombinant human (rh), bovine, and equine CuZn SODs, as well as rh and E. coli Mn SODs, were studied before use with respect to specific activity and purity (HPLC, SDS-PAGE electrophoresis). Fluorescein isothiocyanate was covalently conjugated to the various SODs for study with high-resolution confocal scanning laser microscopy. Superoxide production by monocytes and neutrophils was measured with the cytochrome c assay. RESULTS: As expected from our experiments with flow cytometry, only rare neutrophils were labeled with FITC-SOD, even with the longest incubation time of 3 hr and the highest dose of 1500 units/ml. In addition, they showed a localized fluorescence pattern that was quite different from the diffuse punctate fluorescence pattern of monocytes. Lymphocytes were not labeled at all. The rapid binding to the cellular surface of monocytes was confirmed, and even after 5 min of preincubation, FITC-SOD was found on a small percentage of monocytes. This was correlated with a reduction in superoxide release after phorbolmyristate acetate (PMA) stimulation by 40%. An interesting finding was the perinuclear accumulation of the penetrated SOD after the longest pretreatment of 3 hr, suggesting a barrier against further progression. Indeed, through confocal microscopy we were able to exclude any fluorescence at the nuclear level. While the fluorescence labeling patterns and the kinetics of penetration were quite similar for bovine, equine, and rh CuZn SOD, the Mn SODs showed poor labeling, correlated with a weak inhibitory effect on cytochrome c reduction, which was not statistically significant. CONCLUSIONS: The rapid binding of native CuZn SODs on the surface of monocytes, leading to reduced superoxide release by these cells, explains the observation that beneficial effects of injected SOD lasted for months despite rapid clearance of the enzyme from the bloodstream, according to pharmacodynamic studies. The preferential binding to monocytes, in contrast to neutrophils, may play a role in chronic inflammatory diseases in which the monocytes are in an activated state. The differences in binding capacity between CuZn SODs and Mn SODs, correlated with different inhibitory effects of superoxide production by monocytes, may also have therapeutic significance.     

Sensitive detection of histamine using fluorescently labeled oxido-reductases

A detection scheme is described by which the histamine contents of biological samples can be established. The scheme is based on the use of methylamine dehydrogenase (MADH) which converts primary amines into the corresponding aldehydes and ammonia. The generated reducing equivalents are subsequently transferred to the physiological partner of MADH, amicyanin, which thereby is converted from the oxidized blue-colored form into the reduced colorless form. The change in absorption is detected by monitoring the fluorescence of a covalently attached Cy5 dye label whose fluorescence is (partly) quenched by Förster resonance energy transfer (FRET) to the Cu-site of the amicyanin. The quenching efficiency and, thereby, the label fluorescence, depends on the oxidation state of the amicyanin. When adding histamine to the assay mixture the proportionality between the substrate concentration and the observed rate of the fluorescence increase has enabled this assay as a sensor method with high sensitivity. The MADH and amicyanin composition can be tuned so that the sensor can be adapted over a broad range of histamine concentrations (13 nM–225 μM). The lowest concentration detected so far is 13 nM of histamine. The sensor retained its linearity up to 225 μM with a coefficient of variation of 11% for 10 measurements of 100 nM histamine in a 100 μL sample volume. The use of a label fluorescing around 660 nm helps circumventing the interference from background fluorescence in biological samples. The sensor has been tested to detect histamine in biological fluids such as fish extracts and blood serum.     

Detection of protein oxidation in rat-1 fibroblasts by fluorescently labeled tyramine.

Oxidative damage to proteins has been postulated as a major cause of various degenerative diseases including the loss of functional capacity during aging. A prominent target for oxidation by reactive oxygen species (ROS) is the tyrosine residue. Here we present a highly sensitive method for the detection of tyrosyl radical formation in cells. The method is based on the fluorescein-labeled tyrosine analogue, tyramine, which upon oxidation may couple to proteins carrying a tyrosyl radical. Coupling of the probe (denoted TyrFluo) to standard proteins could be induced by generating ROS with horseradish peroxidase/hydrogen peroxide, SIN-1 or with peroxides (cumene or hydrogen peroxide) in combination with a transition metal. TyrFluo added to rat-1 fibroblasts remained outside the cell, whereas the acetylated form (acetylTyrFluo) was membrane-permeable and accumulated in the cell. Exposure of the cells to oxidative stress in the presence of either TyrFluo or acetylTyrFluo gave a cellular labeling characteristic for each probe. Western blot analysis confirmed that each probe labeled a specific set of proteins. This new method for the detection of ROS-induced oxidation of proteins may mimic the tendency of oxidized proteins to form dityrosine bonds.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.     

Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells

Microtubule-associated proteins (MAPs) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (I-AF). The modified MAPs (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAPs were incorporated predominantly, if not exclusively, into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial, and fibroblastic) of diverse origin (man, mouse, chicken, and rat kangaroo). Mobility measurements of the microinjected AF-MAPs using the method of fluorescence-photobleaching recovery (FPR) revealed two populations of AF-MAPs with distinct dynamic properties: One fraction represents the soluble pool of MAPs and is mobile with a diffusion coefficient of D = 3 X 10(-9) cm2/s. The other fraction of MAPs is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of approximately 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAPs from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAPs can associate in vivo with microtubules of diverse cell types and that treadmilling of MAP2-containing microtubules in vivo, if it exists, is slower than 4 micron/h.     

Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils

Myosin subfragment 1 (S1) can be specifically modified at Lys-553 with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) (Bertrand, R., J. Derancourt, and R. Kassab. 1995. Biochemistry. 34:9500-9507), and solvent quenching of FHS-S1 with iodide has been shown to be sensitive to actin binding at low ionic strength (MacLean, Chrin, and Berger, 2000. Biophys. J. 000-000). In order to extend these results and examine the fraction of actin-bound myosin heads within the myofilament lattice during calcium activation, we have modified skeletal muscle myofibrils, mildly cross-linked with EDC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) to prevent shortening, with FHS. The myosin heavy chain appears to be the predominant site of labeling, and the iodide quenching patterns are consistent with those obtained for myosin S1 in solution, suggesting that Lys-553 is indeed the primary site of FHS incorporation in skeletal muscle myofibrils. The iodide quenching results from calcium-activated FHS-myofibrils indicate that during isometric contraction 29% of the myosin heads are strongly bound to actin within the myofilament lattice at low ionic strength. These results suggest that myosin can be specifically modified with FHS in more complex and physiologically relevant preparations, allowing the real time examination of cross-bridge interactions with actin in in vitro motility assays and during isometric and isotonic contractions within single muscle fibers.     

Binding of protein kinase substrates by fluorescently labeled calmodulin

A synthetic protein kinase substrate, PRO-LEU-SER-ARG-THR-LEU-SER-VAL-SER-SER-NH₂, undergoes calcium-dependent binding by calmodulin. Phosphorylation of the peptide decreases its affinity for calmodulin with the dissociation constant increasing from 2.4 to $\text{ca}$. 7 mM. The results are consistent with the suggestion that calmodulin and the cAMP-dependent protein kinase can act on common recognition sequences.     

Synthesis and biophysical studies on fluorescently labeled oligodeoxyribonucleotides

Two highly fluorescent compounds, viz. 6-(6-isobutyrylamino-1,3-dioxo-1 H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid and 6-(6-dimethylamino-1,3-dioxo-1 H,3H-benzo[de]isoqu-inolin-2-yl)-hexanoic acid have been synthesized, characterized, and attached to 12-mer oligodeoxyribonucleotides at their 5'-end using suitable linker molecule. These labeled oligodeoxyribonucleotides have shown appreciable fluorescence even at 0.0019 microM concentrations. Thermal denaturation studies have shown comparatively higher Tm values when oligodeoxyribonucleotides are labeled. These labeled oligodeoxyribonucleotides have been purified on RP-HPLC utilizing their hydrophobicity and on polyacrylamide gel because of their easy detection due to fluorescence.     

Lectin affinity electrophoresis for the separation of fluorescently labeled sugar derivatives.

Lectin affinity electrophoresis was applied to the separation of charged, fluorescent conjugates of disaccharides. Four fluorescent conjugates were prepared by reductive amination of alpha-D-Man-(1----3)-D-Man, alpha-D-Gal-(1----4)-D-Glc, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc in the presence of 7-amino-1,3-naphthalenedisulfonic acid. These charged fluorescent-disaccharide conjugates all have identical molecular weight and in the absence of conconavalin A lectin failed to separate either by agarose or by polyacrylamide gel electrophoresis. In the presence of either free or immobilized concanavalin A, agarose gel electrophoresis and polyacrylamide gel electrophoresis could separate the fluorescent conjugate of alpha-D-Man-(1----3)-D-Man from that of alpha-D-Gal-(1----4)-D-Gal, alpha-D-Gal-(1----6)-D-Glc, and beta-D-Gal-(1----4)-D-Glc.     

Interaction of fluorescently labeled pardaxin and its analogues with lipid bilayers.

Fluorescence measurements were used to monitor the interaction of the neurotoxin pardaxin and its analogues with membranes. Eight peptides were selectively labeled with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl, either at their N-terminal or at their C-terminal. No detectable changes in membrane permeability or hemolytic activity were observed upon modification. Upon the titration of solutions containing the different peptides with small unilamellar vesicles, the fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled pardaxin and its analogues, but not those of control peptides, displayed blue shifts in addition to enhanced intensities upon relocation of the probe to a more apolar environment. The results revealed that the N terminus of pardaxin is buried within the lipid bilayer while the C terminus is located at the bilayer's surface. Binding isotherms were obtained from the observed increases in the fluorescence emission yields, from which surface partition constants, in the range of 10(4) M-1, were in turn derived. The existence of an aggregation process was suggested by the shape of the binding isotherms. Furthermore, the results show good correlation between the incidence of aggregation and the ability of the different analogues to induce the release of relatively large molecules from vesicles. As such, our results suggest that the mechanism of pore formation employed by pardaxin and its analogues could be described by the "barrel stave" model.     

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.     

The role of different P-glycoproteins in hepatobiliary secretion of fluorescently labeled short-chain phospholipids.

Class III P-glycoproteins (Pgps) mediate biliary phosphatidylcholine (PC) secretion. Recent findings that class I P-glycoproteins are able to transport several short-chain phospholipid analogues raises questions about the role of these Pgps in physiological lipid transport. We investigated the biliary secretion of C6-7-nitro-2,1, 3-benzoxadiazol-4-yl (NBD)-labeled ceramide and its metabolites in Mdr1a/b and Mdr2 knockout mice compared to control mice. Biliary secretion of these NBD-lipids was unaffected in Mdr1a/b -/- mice. Thus neither Mdr1a nor Mdr1b Pgp mediates biliary secretion of these lipids. In contrast, secretion of all three NBD-labeled short-chain phospholipids was significantly reduced in Mdr2 -/- mice. As in vitro studies revealed that Mdr2 Pgp is not able to translocate these lipid analogues, we hypothesized that Mdr2 -/- mice had a reduced PC content of the exoplasmic canalicular membrane leaflet so that extraction of the short-chain lipid probes from this membrane by canalicular bile salts was impaired. To investigate this possibility we studied the bile salt-mediated extraction of natural sphingomyelin (SM) and NBD-labeled short-chain SM from small unilamellar vesicles of different lipid composition. Natural SM could be extracted by the bile salt tauroursodeoxycholate from vesicles containing PC, cholesterol (CHOL), and SM (1:2:2) but not from vesicles containing only SM and CHOL (3:2). NBD-labeled short-chain SM could be extracted from vesicles containing PC while its extraction from pure SM:CHOL vesicles was reduced by 65%.These data confirm that the efficiency of NBD-SM extraction depends on the lipid composition and suggest that the canalicular membrane outer leaflet of Mdr2 -/- mice has a reduced PC content.     

Phagotrophy of fluorescently labeled bacteria by an oceanic phytoplankter

Using fluorescently-labeled bacteria and detection by flow cytometry and epifluorescence microscopy, we demonstrate inducible mixotrophy in a marine photosynthetic flagellate, Ochromonas sp. (class Chrysophyceae). Phagotrophic uptake of bacteria increases under conditions of low or limiting light and nutrients, but deceases in periods of prolonged darkness; sustained phagotrophy may require light. In addition, this alga appears to discriminate between and preferentially ingest different types of bacteria. Although this clone is primarily photosynthetic, phagotrophy contributes to its nutrition, especially when light or nutrients limit photosynthesis.Correspondence to: M.D. Keller