Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.     

Na+-Ca2+ exchange in the axolemma-rich membrane vesicle preparations from the walking-leg nerves of the American lobster

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.     

Fluorescence microscopical visualization of rat mast cells by means of fluoresceine isothiocyanate labeled protamine sulfate.

Using fluoresceine isothiocyanate labeled protamine sulfate it is possible to stain rat mast cell fractions of isolated periotoneal cells in a selective way. The cells are not degranulated, though they have lost their histamine completely. The method shows the dissociation of the mediator release and the extrusion of the granules.     

Synthesis of fluorescent oligosaccharides for covalent attachment to living cells

Asparagine-linked oligosaccharides were liberated from glycoproteins by hydrazinolysis. The treatment resulted in de-N-acetylation of the amino sugars. After isolation of the oligosaccharides free amino groups were labeled with fluorescein isothiocyanate and remaining amino groups reacetylated. The fluorescent oligosaccharides were used to label living cells. They were converted to hydrazine derivatives and covalently attached to cell surface oligosaccharides, which had been treated with periodate or neuraminidase and galactose oxidase. This enabled the visualization of the attached oligosaccharides at the external aspect of the plasma membrane by fluorescence microscopy.     

An immunofluorescent study of post-segregational gene action in rats

Immunofluorescent analysis of the post-segregation effect in mice was carried out. Congenic strains of mice were used. Epididymal sperm of F1 hybrids was studied by means of the indirect method of labelling antibodies with fluoresceine isothiocyanate. The data obtained indicate the possibility of post--segregation effect in mammalian gametes and the possibility of exploration of immunogenetic method for alteration of Mendel's natural correlation in mammals.     

Indirect hemagglutination test at the ultrastructural level]

A study on ultra-thin sections was made of the preparations of agglutinate produced during the reaction of the immunoglobulin erythrocytic diagnostic agent with dry corpuscular Rickettsia prowazeki antigen, fluoresceine isothiocyanate labeled, and also SRBC used for the preparation of the diagnostic agent after formalinization, tannin treatment, sensitization with hyperimmune horse serum immunoglobulins and lyophilization, respectively. Formalin and tannin treatment of erythrocytes failed to be reflected on the ultrastructure of their cellular membranes; the treatment with hemosensitin was accompanied by the appearance of spheroid protrusions of the erythrocyte cytoplasmic membrane with the preservation of its three-layer structure. Specific interaction of sensitized erythrocytes with the antigen corpuscles was expressed morphologically in their apposition or connection through a gap of 20--30 nm.     

Distribution of terminal sugar residues in the testis of the spotted ray Torpedo marmorata

Lectins represent a class of proteins/glycoproteins binding specifically to terminal sugar residues. The present investigation aims to identify lectin-binding sites in testis of Torpedo marmorata. Using a panel of lectins coupled with fluoresceine isothiocyanate, we demonstrated that germ and somatic cells present in Torpedo testis contain glycoconjugates, whose distribution at the level of the surface, the cytoplasm and the nucleus changes during germ cell differentiation. Moreover our observations demonstrate that the germ cells undergoing apoptosis (Prisco et al., 2003a: Mol Reprod Dev 64:341-348) overexpress a residual sugar recognised by WFA lectin that can be considered a specific marker for apoptotic germ cells. Finally, our results indicate that there is a progressive increase in glycosilation during spermatogenesis, especially at the level of the acrosome in the spermatocyte-spermatid step, and that Leydig cells are differently stained in relation to the spermatogenetic cycle.     

Investigations on the use of fluorescence dyes for labeling dendrimers: cytotoxicity, accumulation kinetics, and intracellular distribution

Fluorescent dyes (e.g., dansyl, fluoresceine isothiocyanate, or naphthalimide groups) are widely used as markers to study biological properties of drugs. In order to evaluate possible mediated cytotoxicity, we attached three molecules each to 1,3,5-tris(3-propylamino)benzene initially synthesized as core molecule for the design of dendrimers. Cytotoxic effects were only observed for the NO(2)-substituted naphthalimide conjugate. The intracellular distribution was visualized via confocal fluorescence microscopy and pointed to an accumulation in the endosome or nucleus, dependent on the cell line used.     

Surface polypeptides of the cultured Chinese hamster ovary cell.

The organization of the plasma membrane of logarithmically growing Chinese hamster ovary (CHO) suspension cells has been probed using surface label techniques in conjunction with subcellular fractionation and sodium dodecyl sulfate gel electrophoresis. Five components of apparent molecular weights 137,000, 121,000, 97,000, 67,000, and 57,000 have been shown to be exposed at the outer surface of the cell. These components fully meet the criteria of being (a) reactive with two or more surface label reagents, (b) enriched in a purified plasma membrane fraction, and (c) sensitive to proteolytic digestion of intact cells. Three other components of molecular weights 200,000, 44,000 and 30,000 are also reactive with certain surface label reagents, but fail to meet other criteria for cell surface components. Two polypeptides of molecular weights 180,000 and 37,000 are substantially enriched in the plasma membrane fraction, but are unreactive with surface label reagents. The organization of the CHO cell membrane and the applicability of surface label techniques to cultured cell systems are discussed.     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

Functionalized membrane supports for covalent protein microsequence analysis

Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).     

Cell surface glycoproteins of CHO cells. II. Surface distribution and pathway of internalization

The surface distribution and pathway for internalization of the major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent trinitrobenzenesulfonate. Molecules, haptenized with trinitrophenol groups, the majority of which are in a group of high molecular weight acidic glycoproteins (HMWAG), were labelled at 4 degrees C with anti-dinitrophenol immunoglobulins coupled to fluorescein isothiocyanate (FITC), horseradish peroxidase, or colloidal gold and either immediately fixed for mapping their distribution or followed intracellularly after warming to allow endocytosis to proceed. The distribution of label on the CHO cell surface was non-random with a large proportion arranged in clusters from 100 to 300 nm in diameter. Antibody label was concentrated heavily on microvilli, and about 10% of the molecules were always associated with clathrin-coated pits. Upon warming the cells to 37 degrees C, HMWAG were internalized immediately into smooth-membraned tubules (less than 80 nm luminal diameter) that appeared to connect with vesicles (less than 300 nm luminal diameter) located in the cortical cytoplasm. By 60 min, labelled antibody was located within larger vesicles (greater than 300 nm luminal diameter) that had a morphology characteristic of multivesicular bodies and not lysosomes. There was no evidence for entry of labelled molecules into either electron-dense, secondary lysosomes or into the Golgi cisternae, suggesting that neither compartment is involved in the major pathway of cell surface endocytosis. Our results are consistent with the view that the majority of plasma membrane protein are internalized as small discrete domains by a pathway very similar to that described by others for adsorptive endocytosis.     

Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes.

The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+-ATPase-specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+-ATPase with a large variety of different parameters.     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.     

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.     

Surface modification of polycaprolactone membrane via aminolysis and biomacromolecule immobilization for promoting cytocompatibility of human endothelial cells

Amino groups were covalently introduced onto a polycaprolactone (PCL) surface by the reaction between 1,6-hexanediamine and the ester groups of PCL. The occurrence of the aminolysis and the introduction of free NH(2) groups were verified qualitatively by fluorescence spectroscopy, where rhodamine B isothiocyanate was employed to label NH(2) groups, and quantitatively by absorbance spectroscopy, where ninhydrin was used to react with NH(2) to generate a blue product. Due to the presence of deep pores on the PCL membrane, the aminolysis reaction could penetrate as deep as 50 microm to yield NH(2) density as high as 2 x 10(-7) mol/cm(2). By use of the NH(2) groups as active sites, biocompatible macromolecules such as gelatin, chitosan, or collagen were further immobilized on the aminolyzed PCL membrane via a cross-linking agent, glutaraldehyde. X-ray photoelectron spectroscopy (XPS) and surface wettability measurements confirmed the coupling of the biomacromolecules. The endothelial cell culture proved that the cytocompatibility of the aminolyzed PCL was improved slightly regardless of the NH(2) amount on the surface. After immobilization of the biomacromolecules, however, the cell attachment and proliferation ratios were obviously improved and the cells showed a similar morphology to those on tissue culture polystyrene. Measurement of the von Willebrand factor (vWF) secreted by these endothelial cells (ECs) verified the endothelial function. Hence, a better EC-compatible PCL was produced.     

A spin-label study of the viscosity profile of sarcoplasmic reticular vesicles.

Sarcoplasmic reticular vesicles were prepared from both lobster and rabbit muscle. A variety of water-soluble, lipid-soluble, and alkylating spin labels were used to treat the sarcoplasmic reticular vesicles. All spin label analyses were carried out with and without NiCl₂. Nickel is used to remove spin label signal originating from outside of, on the surface of, or localized in the outermost part of the outer bilayer half of the sarcoplasmic reticular membrane.We conclude that the hydrocarbon portion of sarcoplasmic reticular vesicles has symmetry in regard to the physical properties that limit spin label motion; however, we find that the membrane interface limits spin label motion more on the inner surface than the outer surface and that the trapped aqueous volume which is sampled by water soluble spin labels inside the vesicle enclosure limits spin label motion much more than the average aqueous medium outside.     

Membrane shuttle between plasma membrane, phagosomes, and pinosomes in Dictyostelium discoideum amoeboid cells

The intracellular redistribution of membrane internalized during endocytosis was studied quantitatively by a biochemical approach and by a morphometric analysis of autoradiographs in electron microscopy. Plasma membrane glycoconjugates, enzymatically labelled with radioactive galactose, were used as a membrane marker. In cells labelled at their surface either before or after the phagocytotic uptake of latex beads, subsequent endocytosis led to a redistribution of label between the plasma membrane and endosomal membranes until a steady-state was reached after about 1 h with 43% of the label on the plasma membrane. The steady-state resulted when all participating membranes carried the same surface density of label. During phagocytosis or pinocytosis the equivalent of the plasma membrane was internalized and recycled once every 20 min or 40 min, respectively. Compared to this rate a very rapid and complete mixing of membranes was observed between newly formed phagosomes and preexisting digestive vacuoles or between newly formed pinosomes and preexisting phagosomes. Due to this rapid mixing, the membranes enclosing undigestible latex beads remained fully linked to the shuttle of membrane to and from the cell surface.     

Fluorescent labeling of ursolic acid with FITC for investigation of its cytotoxic activity using confocal microscopy

Fluorescent labeling is a widely-used approach in the study of intracellular processes. This method is becoming increasingly popular for studying small bioactive molecules of natural origin; it allows us to estimate the vital intracellular changes which occur under their influence. We propose a new approach for visualization of the intracellular distribution of triterpene acids, based on fluorescent labeling by fluoresceine isothiocyanate. As a model compound we took the most widely-used and best-studied acid in the ursane series – ursolic acid, as this enabled us to compare the results obtained during our research with the available data, in order to evaluate the validity of the proposed method. Experimental tracing of the dynamics of penetration and distribution of the labeled ursolic acid has shown that when the acid enters the cell, it initially localizes on the inner membranes where the predicted target Akt1/protein kinase B – a protein that inhibits apoptosis – is located.     

Uptake and efflux of fluoresceine inAnacystis nidulans

InAnacystis nidulans uptake and efflux kinetics of fluoresceine were studied. It could be concluded that the distribution of fluoresceine depends on the pH_c of the cells. The comparison of different fluoresceine accumulation levels and flux equilibria allows conclusions regarding changes of the pH_c.