Cytochemical study of concanavalin A binding sites and their mobility in normal, cystic fibrosis, and SV40 transformed human fibroblasts in vitro

Concanavalin A (Con A) binding sites and their mobility were studied by peroxidase (Po) and ferritin labeling techniques in normal and SV40 transformed human fibroblasts. Binding sites were visualized either as osmium black of 3'3-diaminobenzidine (DAB) reactions or as ferritin particles. DAB reaction products were localized at the external surface of the plasma membrane and in some multivesicular bodies of fixed cells. The labeling was continuous in normal and SV40 transformed human fibroblasts. When living cells were treated with Con A-Po at 4 degrees C and incubated at 37 degrees C, both normal and transformed cells showed remarkable changes. The foci of membrane indentations (caps or patches) are formed on the cell surface. Many labeled internalized vacuoles and vesicles appeared within the cytoplasm and in close proximity to the Golgi region of all cell types. The cellular changes occurred more quickly in transformed cells than in normal cells. It is concluded that normal cells do cap under certain conditions and that the plasma membranes of transformed cells are more fluid than those of normal cells.     

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.     

Prevention by retinoic acid of anionic site redistribution on the surface of cultured human sarcoma cells

The distribution of cell surface negatively-charged macromolecules was determined electron microscopically on untreated and on retinoic acid (RA)-treated cultured human osteosarcoma Hs791 and chondrosarcoma Hs705 cells using cationized ferritin (CF), an electron-dense marker of anionic sites. Labeling on the surface of prefixed cells was continuous and uniform whether they were grown in the absence or presence of RA. In contrast, CF distribution on unfixed cells was markedly affected by RA; CF labeling of untreated cells occurred in patches and clusters whereas the label on RA-treated cells was continuous, as on prefixed cells. CF labeling of unfixed cells decreased considerably after incubation of the cells either with hyaluronidase or neuraminidase. There was also a reduction in patching and clustering. Changes induced by RA in the apparent membrane microviscosity, in neuraminidase-releasable sialic acid, or in transglutaminase activity could not be related to the effect of RA on CF-induced anionic site redistribution since these characteristics were modulated differently in the two cell lines. In contrast, RA increased the sialylation of specific cell surface membrane glycoproteins on both cell types. These results suggest that RA prevents redistribution of cell surface sialoglycoconjugates and glycosaminoglycans by CF. This effect may be the result of increased sialylation of specific surface components and may be related causally to the suppression of the transformed phenotype in the sarcoma cells.     

Surface charge of trypanosoma cruzi. Binding of cationized ferritin and measurement of cellular electrophoretic mobility.

The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 micrometer-s-1-V-1-cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from staionary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.     

Leishmania braziliensis: cell surface differences in promastigotes of pathogenic and nonpathogenic strains

A comparative study of cell surface characteristics of pathogenic and nonpathogenic promastigotes of Leishmania braziliensis, NR and LBY strains, respectively, was carried out by means of concanavalin A agglutination and labeling with concanavalin A-fluorescein isothiocyanate, concanavalin A-ferritin, and cationized ferritin. Cytochemical examination showed cell surface differences in lectin receptors and negative charge moieties in the two strains of L. braziliensis. The pathogenic NR strain agglutinated with low concentrations of concanavalin A and presented abundant lectin-binding and cationized ferritin-binding surface labeling. The nonpathogenic LBY strain neither agglutinated when incubated with concanavalin A, bound lectins, or cationized ferritin at the cell surface.     

Surface Charge of Trypanosoma cruzi. Binding of Cationized Ferritin and Measurement of Cellular Electrophoretic Mobility*

SYNOPSIS The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 μm^.s^-1.V^-1.cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from stationary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.     

Endocytosis of cationized ferritin in human resting peripheral blood by T-lymphocytes

Summary We have examined the binding and internalization of cationized ferritin in T-lymphocytes of human peripheral blood, as a model for resting cells. After 30 min of incubation only 8% of endocytotic vesicles contain cationized ferritin. T-cells internalie the equivalent of their entire surface area in approximately 54 h, a longer time than is required by non-resting cells such as PHA-stimulated human lymphocytes. These tracer experiments suggest that the endocytosis of cationized ferritin by T-lymphocytes follows a lysosome pathway similar to that described for other cell types.     

Effect of agents disrupting microtubules on the distribution of receptors on the surface of cultured cells]

Substrate-attached normal mouse fibroblasts, transformed mouse fibroblasts (L-strain) and epithelial cells (MPTR strain) were incubated with two ligands that are cross-linking different group of the surface receptors: concanavalin A and cationic ferritin. Surface-attached ligands were revealed by the indirect immunofluorescent methods. The incubation of control cells with these ligands induced a patching of corresponding surface receptors, and a clearing of these receptors from the surface zones located on the lamellar cytoplasm near the cell edges actively protruding pseudopodia. Effects of three antitubulins (colcemid, colchicine and vinblastin) on the ligand-induced redistribution of receptors were examined and compared with the previously described effects of these drugs on the distribution of active cell edges.     

Differences in density of Concanavalin A-binding sites due to differences in surface morphology of suspended normal and transformed 3T3 fibroblasts.

Calculations of the density of Concanavalin A (Con A)-binding sites on normal and transformed fibroblasts have, as yet, been based on the unproven assumption that suspended cells are smooth spheres. We studied the surface morphology of suspended normal and transformed fibroblasts with scanning and transmission electron microscopes, and found a large difference in surface morphology between suspended normal and transformed 3T3 cells. When this difference in surface morphology was taken into account, the estimated cell surface area of normal 3T3 cells was approximately seven times larger than that of transformed 3T3 cells. Since equal numbers of 3H-Con A molecules are bound on normal and transformed cells, the density of Con A-binding sites is approximately seven times greater on transformed than on normal 3T3 cells. The difference in density of Con A-binding sites between normal and transformed fibroblasts might be sufficient to explain the difference in agglutination response, as originally suggested by Burger, and may also be the cause of the different degrees of clustering of Con A-binding sites on the plasma membrane of these cells.     

Effects of lysolecithin on the surface properties of human erythrocytes

Human red blood cells (RBCs), transformed by incubation with the amphiphatic compound lysolecithin from their normal discocyte shape into echinocytes, have increased rates of agglutination in the presence of either poly- -lysine (PLL) or soybean agglutinin (SBA). Removal of lysolecithin by washing caused a reversal of shape back to the discocyte configuration and a lowering of agglutination rates. Methochlorpromazine, another amphiphatic echinocytogenic substance produced a similar increase in agglutination rates, suggesting that increased agglutinability may be a general property of echinocytes. Lysolecithin treatment of RBCs caused a decrease in the binding of cationized ferritin (CF) particles/μm² of RBC surface. The decrease in CF binding is due to a rearrangement of negative charge bearing molecules on the RBC surface rather than shedding of charged groups. These observations support the hypothesis that integral membrane proteins which bear negative charges and receptors are associated with a cytoskeleton within the red cell. Alterations in cell shape which result in distortion of the cytoskeleton may cause a redistribution of integral membrane proteins which bear charged groups at the RBC surface.     

Regeneration of plasmalemma and surface properties in macrophages

The regeneration of surface anionic groups in mouse peritoneal macrophages was investigated by electron microscopy, using cationized ferritin (CF) as a tool for the localization and evaluation of negative charge density on the cell surface. In vitro interaction of living macrophages with CF resulted in removal of most anionic groups, either by concentration of their receptor sites to a part of the membrane which is subsequently internalized, or by detachment of the aggregated label from the surface. After incubation of macrophages lacking surface anionic groups in tissue culture medium without the ligand, regeneration of the binding capacity for CF took place within 3 h. The first regenerated parts of the membrane can be visualized within 1 h on the upper part of the adherent cells; there is a discontinuous coating of ferritin, with the lateral regions of the plasmalemma free of label. The attached CF particles on the regenerated membrane are closer to the membrane and their density is considerably higher than on the normal control macrophages. The results indicate that the turnover of the plasmalemma is regional and not dispersed; the mechanism involved is insertion of membrane patches into the pre-existing plasma membrane.     

The binding of cationized ferritin at the surfaces of ehrlich ascites tumor cells: the effect of pH and glutaraldehyde fixation.

The densities of cationized ferritin (CF) particles binding to the surfaces of cultured Ehrlich ascites tumor cells were determined at pH 7.4, where the ferritin stain was applied either prior to or following glutaraldehyde fixation. The densities were also determined with CF adjusted to pH 1.9 and applied after fixation. For all fixed samples there was a higher density of particles bound to microvilli than to the spaces between them. Treatment with neuraminidase removed more particles from microvilli than from the inter-microvillus spaces, but did not reduce the levels of binding to the same value. When cationized ferritin is applied prior to fixation, an aggregation of the CF particles at the cell surface was observed, with the internalization of some clusters. This effect was independent of neuraminidase treatment.     

Anionic sites on the envelope of Salmonella typhimurium mapped with cationized ferritin

Binding of either ferritin (F) or cationized ferritin (CF) was employed to indicate the surface charge of the envelope of mainly two Salmonella typhimurium strains (395 MR10, a Rd-mutant, and LT2-M1, a UDP-galactose-4-epimerase-less mutant). Lowering the pH from 7 to 4 decreased binding of CF, but increased binding of F. At low concentrations, the distribution of CF on S. typhimurium 395 MR10 was in general random, with individual ferritin molecules often forming clusters of two or three particles. At ionic strengths of 0.25M NaCl, ferritin produced distinctive, larger clusters at relatively few sites (10-50/cell). Addition of galactose to cultures of growing S. typhimurium, LT2-M1 reduced the binding of CF in 1-10 min, and numerous ferritin-free areas became visible. Possibly this is caused by a pluri-focal reduction in the negative cell surface charge that was generated at the multiple sites of export of new, smooth-type lipopolysaccharide, which either exhibits lesser charge or masks a preexisting surface charge. Dividing cells may show unequal charges on the prospective daughter cells, and the difference in the capacity for ferritin adsorption of both daughter cells is sharply separated at the division site.     

Changes in the distribution of anionic constituents in secretory granules of mouse pancreatic acinar cells after pilocarpine-induced degranulation.

We used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. After pilocarpine-stimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (approximately 43.0 gold particles/microm2) compared to the 8-hr mature granules group (approximately 64.6 gold particles/microm2). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.     

Anionic sites on the envelope ofSalmonella typhimurium mapped with cationized ferritin

Binding of either ferritin (F) or cationized ferritin (CF) was employed to indicate the surface charge of the envelope of mainly twoSalmonella typhimurium strains (395 MR10, a Rd-mutant, and LT2-M1, a UDP-galactose-4-epimerase-less mutant). Lowering the pH from 7 to 4 decreased binding of CF, but increased binding of F. At low concentrations, the distribution of CF onS. typhimurium 395 MR10 was in general random, with individual ferritin molecules often forming clusters of two or three particles. At ionic strengths of 0.25M NaCl, ferritin produced distinctive, larger clusters at relatively few sites (10–50/cell). Addition of galactose to cultures of growingS. typhimurium, LT2-M1 reduced the binding of CF in 1–10 min, and numerous ferritinfree areas became visible. Possibly this is caused by a pluri-focal reduction in the negative cell surface charge that was generated at the multiple sites of export of new, smooth-type lipopolysaccharide, which either exhibits lesser charge or masks a preexisting surface charge. Dividing cells may show unequal charges on the prospective daughter cells, and the difference in the capacity for ferritin adsorption of both daughter cells is sharply separated at the division site.     

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates

Synopsis Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.     

Evidence for a decreased membrane recycling in the cells of renal proximal tubules exposed to high concentrations of ferritin

Summary The present study was performed to investigate whether membrane recycling via the dense apical tubules in cells of renal proximal tubules could be modified after exposure to large amounts of cationized ferritin. Proximal tubules in the rat kidney were microinfused in vivo with cationized ferritin for 10 or 30 min and then fixed with glutaraldehyde by microinfusion, or proximal tubules were microinfused with ferritin for 30 min and then fixed 2 h thereafter. The tubules were processed for electron microscopy, and the surface density and the volume density of the different cell organelles involved in endocytosis were determined by morphometry. The morphometric analyses showed that after loading of the endocytic vesicles with ferritin the surface density of dense apical tubules decreased to about 50% of the original value. However, 2 h later when ferritin had accumulated in the lysosomes the surface density of dense apical tubules had returned to control values. Furthermore, cationized ferritin was virtually absent from the Golgi region, indicating that the Golgi apparatus in these cells does not participate in membrane recycling. In conclusion, the present study shows that membrane recycling in renal proximal tubule cells can in part be inhibited by loading the endocytic vacuoles with ferritin.     

Surface differentiation of hemopoietic cells demonstrated ultrastructurally with cationized ferritin

The ultrastructural cationized ferritin (CF) technique was employed as a probe of the surface binding characteristics of the various cell types present in normal human bone marrow. The number of CF particles per micron length of cell surface were counted and data subjected to statistical analysis. All cells of the bone marrow exhibited CF reactivity. The extent of labeling was cell specific and could be related to the stage of maturation of the cells in a given lineage. In the neutrophilic series, myeloblasts showed moderate labeling while promyelocytes and myelocytes revealed only minimal binding; CF binding increased sequentially in metamyelocytes, band and segmented neutrophils. Eosinophils and eosinophilic myelocytes showed similar membrane differnetiation patterns while basophils exhibited stronger CF labeling that other granulocytic cells. Lymphocytes were strongly reactive while monocytes and their precursors were moderately labeled with CF. Surface reactivity of developing nucleated erythrocytic cells was similar to that of the lymphocytes. Surface labeling from the proerythroblasts to early normoblasts stage was identical, CF binding increased in the late normoblasts stage and then decreased in the reticulocyte and mature erythrocyte stages. The extent of surface CF reactivity of the marrow cells was markedly different from that obtained with Thorotrast and colloidal iron. Thorotrast and colloidal iron stained the surface of all marrow cell intensely but failed to yield distinctive surface labeling patterns for the differing cell population in bone marrow.     

Phospholipid composition and turnover in normal and SV40 transformed fibroblasts. Effect of cell density]

The phospholipid composition and turnover in normal and in SV 40 transformed hamster fibroblasts were studied. The amount of phospholipid phosphorus relative to protein is lower in transformed hamster fibroblasts than in normal fibroblasts. This amount decreases with increasing cell density until stationary growth is reached. The decrease is largest for the normal fibroblasts. In transformed cells, less sphingomyelin and more diphosphatidyl glycerol are found than in normal cells. The turnover of 32P in sphingomyelin is slower in transformed cells than in normal cells ; the contrary is observed with diphosphatidyl glycerol. On the other hand, in transformed cells, phosphatidyl ethanolamine has a faster turnover than phosphatidyl choline, whereas the contrary is observed in normal cells. Finally, the change to stationary growth slows down the turnover of 32P of all phospholipids, this decrease being more important in transformed cells.     

Anionic sites,fucose residues and class I human leukocyte antigen fate during interaction of Toxoplasma gondii with endothelial cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocyte antigen (HLA) before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containing vacuoles.