Cell Surface Anionic Sites Research Articles

Optic nerve microvessels: a partial molecular definition of cell surface anionic sites

Incorporated in the luminal glycocalyx of vascular endothelia (EC) are negatively charged microdomains (anionic sites). These sites are considered functionally important (a) in their interaction with circulating blood constituents, and (b) as a determinant of vascular permeability. The molecular composition of these EC sites, described for a number of tissues, has demonstrated a heterogeneity dependent on their anatomical location. Luminal anionic sites have not been characterized for EC of optic nerve. Optic nerves were removed from Sprague-Dawley rats previously fixed by vascular perfusion. EC anionic sites were labelled with the probes cationic colloidal gold (CCG) and cationic ferritin (CF), using the pre- and post-embedding techniques, and examined by electron microscopy. The effects of enzyme digestion of ultrathin sections on subsequent CCG labelling were determined using a battery of enzymes in association with the post-embedding technique. CCG labelling was quantified following each enzyme treatment using image analysis software. The biotinylated lectin wheat germ agglutinin (WGA) with streptavidin gold was also used to localize specific monosaccharide residues. The luminal front of intraneural EC showed a uniform labelling with CCG and CF which was greater than on the abluminal surface. Extracellular matrix components and basal laminae were moderately labelled. Digestion of tissue sections with heparitinase and trypsin had no significant effect on subsequent CCG labelling. Proteinase K was less effective than papain but both produced a significant reduction. Neuraminidase almost completely eliminated labelling. CCG binding to the luminal plasma membrane of optic nerve EC can be significantly reduced with proteolytic and glycolytic enzymes. The results demonstrate that sialoglycoproteins principally constitute these luminal EC anionic sites. Biotinylated WGA-streptavidin gold, which detects both N-acetylneuraminic (sialic) acid and N-acetylglucosamine, gave a similar pattern of labelling to CCG alone on the luminal versus abluminal EC fronts. These findings suggest that WGA is binding predominantly to N-acetylneuraminic acid residues since CCG would not label the neutral (uncharged) N-acetylglucosamine.

Anionic site behavior in Leishmania and its role in the parasite—macrophage interaction

The behavior of cationized ferritin (CF) binding sites on the surface of Leishmania mexicana amazonensis (amastigotes, infective and non-infective promastigotes) and their participation in the interaction with macrophages were evaluated. Glutaral-dehyde-fixed parasites treated with CF present a uniform labelling over the whole cell surface. However, living parasites displayed CF patches and caps. Capping was usually seen towards the anterior (flagellated) portion of the cells, where shedding phenomena took place. These processes were inhibited by sodium azide but not by low temperature (4 degrees C). CF treatment of non-infective promastigotes led to an increase in their uptake by macrophages, whereas the uptake of amastigotes or infective promastigotes was not significantly altered. The effect of CF on the parasite surface charge was analyzed by whole-cell microelectrophoresis. The mean electrophoretic mobility (EPM) of non-infective promastigotes was decreased by 26%, while once again the other parasite forms were not significantly affected. Transmission electron microscopy of mouse peritoneal macrophage cultures, fixed after interaction with CF-labelled parasites, revealed that both amastigotes and infective promastigotes quickly removed bound CF. Therefore CF was seen neither in parasite-macrophage attachment areas nor in parasitophorous vacuoles. On the contrary, non-infective promastigote-macrophage attachment areas were remarkably large and preferentially comprised CF-labelled membranes. These results strongly suggest an important participation of cell surface anionic sites in the L. mexicana amazonensis-macrophage interaction.

Calf cardiac valvular endothelial cells in culture: Production of glycosaminoglycans, prostacyclin and fibronectin

To study the roles played by cardiac valvular endothelium in normal and pathologic conditions, we have established and characterized a system of bovine valvular endothelial cells (VEC) in culture. Viable VEC from calf atrioventricular valves were obtained by a non-enzymatic procedure using 3 mM ethylenediamine-tetraacetic acid (EDTA) as dissociating agent. The cells grown in Dulbecco's modified Eagle's medium supplemented with non-essential amino acids, vitamins and 20% fetal calf serum, developed as monolayers of closely apposed polygonal cells which were subcultured for up to seven passages. VEC maintained in culture the general ultrastructure displayed in vivo, expressed von Willebrand factor, presented angiotensin converting enzyme activity and synthesized a rich extracellular matrix. VEC preserved the cell surface anionic sites (detected with cationized ferritin, pI 8.4) and cationic sites (visualized with haemeundecapeptide pI 4.85), and took up, especially by adsorptive endocytosis, albumin-gold conjugate. The cells were coupled by functional communicating (gap) junctions, as demonstrated by microinjection of 6-carboxyfluorescein. VEC in culture produced fibronectin, prostacyclin, hyaluronic acid and heparin-like glycosaminoglycans (identified by electrophoresis, enzyme digestion, and deaminative cleavage of molecules). These properties render cultured VEC a suitable model for investigating their functions and involvement in normal and pathologic heart valves.

Chitosan-colloidal gold complexes as polycationic probes for the detection of anionic sites by transmission and scanning electron microscopy.

A new cationic colloidal gold complex has been developed for ultrastructural localization of cell surface anionic sites by transmission and scanning electron microscopy. The marker is prepared by labelling gold particles of suitable sizes (6 to 70 nm in diameter) with chitosan, a polymer of beta (1----4)-linked D-glucosamine. Using human red blood cells as a model, chitosan-gold complexes were shown to be specific for anionic sites and at pH 2 for sialic acid residues. The binding capacity of complexes of different sizes with carboxymethyl and phosphorylated celluloses was examined as a function of pH and ionic strength. The results indicated that these complexes can be used under acidic conditions as well as in physiological buffers. The complexes were further tested by transmission and scanning electron microscopy in detecting anionic sites on cells of various origins such as Escherichia coli, Lactobacillus maltaromicus, Lactobacillus reuteri, Saccharomyces cerevisiae, Saccharomyces rouxii, Schizosaccharomyces pombe, Fusarium oxysporum, Catharantus roseus.

A simple two-step labeling procedure for ultrastructural localization of cell surface anionic sites.

We propose a new method for ultrastructural localization of cell surface anionic sites. The method consists of sequential interaction of aldehyde-fixed cells with a polycationic reagent, poly-L-lysine (PL), followed by secondary interaction with a negatively charged marker, ferritin. By use of PL of low molecular weight (4000) on aldehyde-pre-fixed red blood cells and macrophages, the reaction resulted in binding of ferritin particles to cell surface anionic sites with a density distribution resembling that of cationized ferritin (CF). The density of the attached ferritin molecules increased in direct correlation with the MW of PL used. The primary PL interaction can be carried out at low pH (less than 2), thus restricting the labeling mainly to membrane-bound sialyl residues.

Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium.

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.

Double labeling study of anionic sites and concanavalin A binding sites in monkey macrophages.

Cationized ferritin (CF) binding, and its effect on the concanavalin A (Con A) binding pattern were studied by the double technique in monkey peritoneal macrophages. CF particles formed clumps and were internalized when cells were incubated at 37 degrees C. Such cells were fixed, and the Con A binding sites were visualized by the Con A-horseradish peroxidase (HRP) method. Using the same specimen, the distribution of CF particles and the Con A-HRP product was observed under an electron microscope. The redistribution and internalization of CF particles did not affect the continuous label of the cell surface Con A binding sites. These observations suggest the independent mobility of cell surface anionic sites and Con A binding sites.

Disappearance of anionic sites from the surface of the rat endometrial epithelium at the time of blastocyst implantation.

Studies have been carried out to analyze possible changes in the distribution of anionic sites on the endometrial epithelium that might play a role in blastocyst implantation. Anionic sites were stained in nonfixed specimens at physiologicalpH and ionicstrengthutilizingpolycationicferritin and were subsequently observed by electronmicroscopy. Experiments were carriedout usingboth in situ and in vitrolabelingconditions, with either pregnant or pseudopregnant rats. There was a general reduction of anionic sitesfrom Days 2-6 of pregnancy or pseudopregnancy untilat Day 6 anionic sites were no longer detected. The results were similar on the mesometrial and antimesometrial sides of the uterus and on presumptive implantation sites(visualizedby pontamine blue staining)and elsewhere on the endometrial epithelium. These findingssuggest that the uterus undergoes a hormonally controlledlossof cellsurface anionic sites,which may be relatedto the abilityof the blastocystto implant about Days 5-6 but not earlier.

Measurement of anionic sites on the surfaces of baby hamster kidney cells using radiolabeled polycationic ferritin

A method is described to determine relative numbers of anionic sites on the surfaces of cells under physiological conditions by binding studies with radiolabeled polycationic ferritin. Labeling of cells by polycationic ferritin occurred very rapidly even at 2°C and was essentially complete within 1 min. At 22°C, a rapid initial phase of labeling was followed by a second, slower binding phase. The interaction of rapidly labeled cell surface anionic sites with polycationic ferritin had a binding constant of 3.6 × 10 6 m −1 (measured at 2°C) and there were about 4 × 10 6 of these sites per cell.

Distribution and movement of anionic cell surface sites in cultured human vascular endothelial cells

Cell surface anionic sites on primary and transformed cultures of human vascular endothelium were studied using cationized ferritin (CF) as an ultrastructural marker. The native distribution of anionic sites on the upper (free) surfaces of cells, fixed in situ with glutaraldehyde, was uniform. Binding of the polycationic ligand, CF, in unfixed cells induced redistribution of anionic sites. Rapid formation of discrete patches of CF particles was followed by reappearance of binding between patches, movement of surface-bound CF into intercellular clefts, and endocytosis, over the next 30 min. Aldehyde-fixed cells, detached from the culture surface, bound CF on both upper and lower surfaces. The distribution and mobility of negatively charged membrane components in vascular endothelium may have relevance for thrombosis, atherogenesis, and vascular permeability.

Redistribution of cell surface anionic sites on hepatoma cells after treatment with Concanavalin A

Chang rat hepatoma ascites cells were treated with Con A and subsequently labeled with cationized ferritin (CF). When ascites cells were treated with Con A, fixed with glutaraldehyde, then labeled with CF, the labeling on the surface of the microvilli and plasma membrane was clustered and irregular. The areas of the cell surface between CF clusters were either lightly labeled or completely free of label. Cells fixed before Con A and CF treatment or cells incubated with Con A and α-methyl- d-mannoside were labeled with a continuous, even layer of ferritin particles. Treatment of ascites cells with cetyl pyridinium chloride before CF labeling resulted in an almost complete absence of ferritin particles from the cell surface.

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.

Localization of sialyl residues on cell surfaces by affinity cytochemistry

The use of the avidin—biotin complex for the specific ultrastructural visualization and evaluation of cell surface sialyl residues was investigated. Circulating red blood cells of various mammalian species and bone marrow cells of the rabbit were treated with sodium meta -periodate and biotin hydrazide in succession, with or without prior treatment of the cells with neuraminidase. The results were compared with the distribution of cell surface anionic sites, estimated by the rate of agglutination with poly- l -lysine and the binding capacity of positively charged colloidal iron and cationized ferritin. A uniform labeling of the erythrocyte surface was obtained with variations between different animal species. Neuraminidase treatment drastically reduced the sensitivity of cells to periodate-induced biotinylation. This method represents a distinct improvement over past approaches, since it can be applied to unfixed cells at physiological pH. An additional feature of this system enabled the topographic resolution of sialic acid residues from the outer dense line of the erythrocyte membrane. A value of 50–70 Å was obtained for human erythrocytes. Developmental and species-dependent modulations in the proximity of sialic acid to the membrane were also observed. The frequency of attached ferritin—avidin conjugates to biotinylated erythroid cells in rabbit bone marrow was found to vary according to the degree of maturation in the erythroid line, corresponding to the previously described variations in colloidal iron binding.

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells.

The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells were studied by utilizing the multivalent ligand, polycationic ferritin, as a visual probe. Our observations revealed that anionic sites are distributed over the entire cell surface, with the highest density of sites being located on cell surface microextensions. Following the initial binding of polycationic ferritin to the surface of unfixed cells, the ligand-bound anionic sites redistributed by migrating from the surface of microextensions to the surface of the cell body. In 20 min, this migration resulted in a total clearing of anionic sites from the surface of microextensions concomitant with the formation of patches of anionic sites on the surface of the cell body. Polycationic ferritin-induced migration and patch formation of anionic sites was not prevented by 2,4-dinitrophenol, N-ethylmaleimide, colchicine, or cytochalasin B. However, the ligand-induced redistribution of cell surface anionic sites was prevented by prefixation of cells with glutaraldehyde.

Heterogeneity of anionic sites at the electrokinetic surfaces of fixed ehrlich ascites tumor cells

The net surface negativity of Ehrlich ascites tumor cells was reduced by treatment with neuraminidase and/or ribonuclease. After fixation in glutaraldehyde, the electrophoretic mobilities of the cells were measured, and their average surface charge densities calculated. Fixed cells were also reacted with net positively charged colloidal iron oxide particles, embedded, cut into sections of known approximate thickness, and examined by electron microscopy. Particle counts per unit area of cell surface, which are thought to indicate coulombic binding to cell surface anionic sites, bore no proportional relationship to the surface density of anionic sites calculated from the electrokinetic data. The two sets of data were best “explained” by postulating that the enzyme-sensitive cell surface anionic sites are arranged in higher-than-average density clusters. On average, 30 RNase-susceptible surface anionic sites were covered by one iron particle (c. 8000 Å 2), compared with 20 neuraminidase-susceptible sites. It was also postulated that the anionic sites not susceptible to these two enzymes, were spread more homogeneously over the cellular electrokinetic surface. This model provides a reasonable explanation for the lack of correlation between observed cell social behaviour, and their electrophoretic mobilities, in terms of the heterogeneous distribution of surface ionogenic groups.

A rapid method for determining the topological distribution of anionic sites on membrane surfaces.

AbstractA method is described for the topological localization of cell surface anionic sites using positively charged colloidal iron hydroxide (Gasic et al., Lab. Invest. 18, 63 [1968]). Purified glutaraldehyde‐fixed plasma cell membranes or glutaraldehyde‐fixed lysed cells are mounted on thin support films and directly stained with colloidal iron at pH 1.8, washed, dried, and then examined by transmission electron microscopy. Using this method the surface anionic residues on rabbit erythrocytes and murine MOPC‐70A myeloma cells are presently in randomly spaced clusters. The distribution of anionic residues on rabbit spermatozoa is discontinuous: the sperm tails almost exclusively bind the colloidal iron in a dense random distribution, while the heads show little or no labeling.