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.     

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 × 10⁶m^?1 (measured at 2°C) and there were about 4 × 10⁶ of these sites per cell.     

CHANGES IN CHARGED GROUPS ON THE CELL SURFACE DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM: AN ELECTRON MICROSCOPIC STUDY

The distribution of charged groups on the surface of Dictyostelium cells and their change during development were examined by electronmicroscopy using cationic and anionic ferritins. The number of anionic sites on the cell surface decreased greatly during the course of development. The whole surface of vegetative cells stained strongly with cationic ferritin (CF). On the other hand, the surface of aggregation-competent cells had fewer negative charges and these were unequally distributed, the surface of the advancing area (lobopodial region) being devoid of anionic sites. The number of anionic sites on the cell surface decreased progressively during further development, and the suface of slug cells did not stain at all with CF. The cell surface did not stain with anionic ferritin at any developmental stage, indicating the absence of detectable cationic sites. The biological significance of these findings is discussed in connection with cell adhesiveness and movement.     

Cationic ferritin binding sites and surface charge densities of transformed cells

Bioelectric surface properties of the high and low tumor-producing cell lines, NCTC 2472 and NCTC 2555, respectively, were determined by cationic ferritin binding and the electrophoretic mobility of intact cells. Measurements of anionic sites were bases on the number of cationic ferritin particles per 0.01 mu 2 that were electronically tagged and counted by an image analyzer. The average particle count was 45 for the control "high" cells and 34 for the control "low" cells. The surface charge densities, expressed as electrostatic units per cm-2 x 10(-13) were 2.34 and 1.18 at 50 V (2 mA) for the "high" and "low" control cells, respectively. Enzymic cleavage of sialic acid and other carbohydrate moieties resulted in up to an 81% reduction in the charge densities and a 57% reduction of the anionic sites of the "high" cells. The electrophoretic mobility of cells with bound cationic ferritin showed that up to 50% of the exposed anionic sites fail to bind cationic ferritin. Preliminary findings on the particle size/distribution by image analysis showed wide ranges in both particle size and interparticle distances that may limit cationic ferritin binding.     

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 distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.     

Nonrandom distribution of sialic acid over the cell surface of bristle- coated endocytic vesicles of the sinusoidal endothelium cells

Previous studies with protein tracers have shown that the luminal surface of the vascular endothelium of the bone marrow is endocytic. The endocytosis occurs through the formation of large bristle-coated vesicles (LCV). The anionic charge distribution in this process was examined at the luminal surface of the endothelial cell, At pH 1.8, colloidal iron (CI), native ferritin, and polycationic ferritin (PCF) are bound by the luminal surface of the endothelial cell, but not at the sites of LCV formation. PCF used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels) revealed LCV binding of this agent in increasing manner from pH 3.5 upwards. PCF binding at low pH (1.8) at the endothelial cell surface was markedly reduced by neuraminidase. Neuraminidase did not reduce PCF binding by the endothelial cell surface nor by the LCV at higher pH levels. It is concluded that the luminal surface of the endothelial cell has exposed sialic acid groups which are absent or significantly diminished at endocytic sites. The free surface of the endothelial cells as well as the sites of endocytosis have, in addition, anionic material with a pKa higher than that of sialic acid (pKa 2.6). These anionic materials may be different at the sites of endocytosis as compared to those present at the free cell surface.     

Preparation of ferritin-avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry.

An improved technique was developed for the unidirectional covalent binding of avidin to ferritin by reductive alkylation. The method is based on the oxidation of sugar moieties on avidin and subsequent coupling to amino groups of ferritin via Schiff's bases followed by reduction with sodium borohydride. The resultant conjugate was used as an ultrastructural marker for the localization of surface receptor sites on biotin-derivatized whole cells. Erythrocytes were treated chemically with sodium meta-periodate and biotin hydrazide in succession. The ferritin-avidin conjugates were used to label the biotin sites either before or after fixation of the cells. The density and distribution of ferritin avidin conjugates on cell surfaces were anlyzed on thin sections and compared with those of cationized ferritin, which were shown to bind anionic sites of the erythrocyte membrane. The extensions of this method for the visualization of other systems is discussed.     

The distribution of anionic sites on the surface of the Golgi complex

The distribution of anionic binding sites has been investigated in the isolated Golgi complex using cationic ferritin. The greatest density of anionic sites occurs on the tubular network and small vesicles, and this binding is accompanied by increased levels of galactosyltransferase activity. The density of anionic sites on the cisternae is less than on the tubules and shows anisotropic distribution, with higher density on the convex surface and lower density on the concave surface. The distribution of anionic sites may reflect the functional activity of the Golgi complex and possibly the interaction or cohesion between cisternae in this organelle.     

Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages

The participation of cell surface anionic sites on the interaction between tachyzoites of Toxoplasma gondii and macrophages and the process of phagosome-lysosome fusion were analyzed using cationized ferritin as a marker of cell surface anionic sites and albumin-colloidal gold as a marker for secondary lysosomes. Incubation of either the macrophages or the parasites with cationized ferritin before the interaction increased the ingestion of parasites by macrophages. Anionic sites of the macrophage's surface, labeled with cationized ferritin before the interaction, were internalized together with untreated parasites. However, after interaction with glutaraldehyde-fixed or specific antibody-coated parasites, the cationized ferritin particles were observed in endocytic vacuoles which did not contain parasites. Macrophages previously labeled with albumin-gold at 37 degrees C, were incubated in the presence of cationized ferritin at 4 degrees C and then incubated with untreated or specific antibody-coated parasites. After interaction with opsonized parasites, the colloidal gold particles were observed in the parasitophorous vacuoles while the cationized ferritin particles were observed in cytoplasmic vesicles. However, when the interaction was carried out with untreated parasites, the parasitophorous vacuoles exhibited ferritin particles while the colloidal gold particles were observed in cytoplasmic vesicles. These observations, in association with studies previously reported, suggest that the state of the parasite surface determines the mechanism of parasite entry into the macrophage, the composition of the membrane lining the parasitophorous vacuole and the ability of lysosomes to fuse with the vacuoles.     

The role of sialated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium.

Changes in the anionic charge distribution at the luminal face of the endothelium of the sinusoids of the bone marrow have been studied at sites of endocytosis by large bristle coated vesicles and at the sites of molecular permeability through diaphragmed fenestrae. The anionic charge distribution has also been studied at the abluminal aspect of these vessels at sites of transmural blood cell passage. Cationic surface markers such as colloidal iron, native ferritin and polycationic ferritin used at low pH, 1.8, and the use of neuraminidase show that the nonmodified endothelial cell surface has exposed sialic acid groups, which are absent at the sites of these functional specializations. Polycationic ferritin binding over a range of pH levels indicates the prsence of another species of anionic materials present at both the nonmodified cell surface and at the sites of the cell surface modifications. This second group of anionic compounds is neuraminidase resistant and has a pKa higher than that of sialic acid (pKa:2.6).     

Asymmetric distribution of anionic sites on rat liver nuclear membranes.

The labelling characteristics of isolated rat liver cell nuclei was studied using polycationized ferritin as an ultrastructural probe for anionic sites. At low concentrations of the marker the nuclear surface was partly labelled eg. at sites of nuclear annuli. At high probe concentrations the entire cytoplasmic surface of the outer nuclear membrane bound ferritin particles. On the other hand, the cisternal surfaces of nuclear membranes could not be labelled although in parallel experiments Concanavalin A-ferritin bound to the cisternal surface of both nuclear membranes indicating free access of ferritin particles to the perinuclear space. The results indicate that nuclear membranes show a distinct vectorial asymmetry in respect to the presence of anionic surface sites.     

Redistribution of surface anionic sites on the luminal front of blood vessel endothelium after interaction with polycationic ligand

The ability of anionic groups on the luminal surface of blood vessels to redistribute by lateral migration under the influence of multivalent ligands was analyzed by electron microscopy, using cationized ferritin (CF). In vitro interaction of blood vessel segments with CF results in rapid aggregation of most anionic sites on the luminal fromt of the endothelium, followed by internalization or detachment of the CF patches, leaving most of the luminal surface devoid of anionic sites. Further incubation of such endothelial cells without CF results in regeneration of binding capacity for the polycationic label. Transport of CF, but not of native ferritin, across the endothelium by vesicle transport, followed by exocytosis of the interiorized CF clusters on the tissue front of the endothelium, was also observed. The possibility that such activities in the blood vessels in vivo may be associated with local changes in the normal distribution of the surface anionic sites as well as in accumulation of debris in the subendothelial layers of the vessels is suggested.     

Correlation of anionic residue mobility at the filopodial plasma membranes with multilayer formation in transformed 3T3 cells

Simian virus 40-transformed 3T3 (SV40-3T3) cells formed multilayers on a Falcon dish and had numerous filopodial projections, some of which intertwined with those of adjacent cells, in contrast to the few projections of their nontransformed counterparts. When these cells were incubated with polycationic ferritin (0.5 mg/ml), ferritin particles, representative of anionic sites, were spread widely on their surfaces at 4 degrees C, while they formed clusters at 37 degrees C, especially on filopodial surface areas opposing adjacent projections in SV40-3T3 cells. These findings demonstrate an increase in the mobility of molecules with anionic residues on filopodial plasma membranes in SV40-3T3 cells, thus suggesting a role for these projections in the formation of multilayered cell aggregates.     

Effects of cationized ferritin and neuraminidase on invasion of cultured cells by Eimeria meleagrimitis sporozoites

Primary turkey kidney cells and Eimeria meleagrimitis sporozoites were treated with cationized ferritin (CF) or neuraminidase ( NANase ), and the effects on the invasion of the cells by the sporozoites were measured. Cultures of host cells pretreated with either compound contained significantly fewer intracellular sporozoites than did control cultures. There was little additive effect if cultures were first treated with NANase and then with CF. In contrast, pretreatment of sporozoites with CF or low concentrations of NANase had no effect on invasion. The inhibition of invasion was apparently due to an interaction between treatment substances and host cell surface rather than to direct effect on the sporozoites. The CF bound to the randomly distributed anionic sites on the surfaces of both host cells and sporozoites and then rapidly aggregated. Sporozoites, probably in the process of invading cells, were invariably found with the conoid in close association with aggregates of CF on the host cell membrane. The CF on the sporozoites was apparently shed before or during invasion because all intracellular sporozoites were completely devoid of the label.     

Effect of colchicine, vinblastine, and cytochalasin B on cell surface anionic sites of Tritrichomonas foetus

Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic site-containing macromolecules located on the cell surface of T. foetus.     

Effects of Cationized Ferritin and Neuraminidase on Invasion of Cultured Cells by Eimeria meleagrimitis Sporozoites

Primary turkey kidney cells and Eimeria meleagrimitis sporozoites were treated with cationized ferritin (CF) or neuraminidase (NANase), and the effects on the invasion of the cells by the sporozoites were measured. Cultures of host cells pretreated with either compound contained significantly fewer intracellular sporozoites than did control cultures. There was little additive effect if cultures were first treated with NANase and then with CF. In contrast, pretreatment of sporozoites with CF or low concentrations of NANase had no effect on invasion. The inhibition of invasion was apparently due to an interaction between treatment substances and host cell surface rather than to direct effect on the sporozoites. The CF bound to the randomly distributed anionic sites on the surfaces of both host cells and sporozoites and then rapidly aggregated. Sporozoites, probably in the process of invading cells, were invariably found with the conoid in close association with aggregates of CF on the host cell membrane. The CF on the sporozoites was apparently shed before or during invasion because all intracellular sporozoites were completely devoid of the label.     

Charged sieving by the basal lamina and the distribution of anionic sites on the external surfaces of fat body cells

The distribution of anionic sites on the basal lamina has been examined with highly cationic ferritin. The penetration of ferritins, with a range of charges from anionic to highly cationic, through the basal lamina into the spaces between fat body cells in an insect is correlated with the charge of the tracer. The anionic sites of the basal lamina may therefore affect the composition of the lymph that bathes the fat body cells. There was more cationic ferritin bound to the plasma membrane reticular reticular system than to the lateral plasma membranes, suggesting that there may be regional differences in surface charge.     

The distribution of cationized ferritin receptors on ciliated epithelial cells of rat trachea

The interaction of tracheal cilia with the biphasic mucus layer covering the surface of the mammalian respiratory tract may be influenced by many cell surface coat components including those having an overall negative charge. In order to assess the distribution of ciliary anionic sites, cationized ferritin (CF) was used to label the surface of rat tracheal epithelium. If pieces of trachea were fixed with 3% glutaraldehyde and treated with CF at low (L) (0.08 mg/ml), medium (M) (0.32 mg/ml PBS), or high (H) (0.64 mg/ml PBS) concentrations, the label was distributed evenly over the entire external surface of the ciliary membrane at all concentrations. Unfixed tracheal tissue was also treated with L, M, and H CF for 1 or 5 min at 4 degrees C in order to minimize lateral redistribution of CF receptors. To ensure accessibility of the cell surface to CF the samples were agitated thoroughly during exposure. Exposure for 1 min to L, M, and H CF resulted in a light binding of ferritin particles on all portions of the ciliary membrane with occasional areas of multilayered binding distributed randomly on the ciliary shaft. When unfixed trachea was treated with CF for 5 min at 4 degrees C, CF binding was similar except heavier and more uniform. In no instance was there any preferential binding of CF to the ciliary tips at any of the concentrations used. Moreover, as indicated by the CF binding pattern at L concentrations, high density negative charges are present over almost the entire surface of the cilium. These results suggest that, unlike the ciliary membrane of other organs such as oviduct, negatively charged cell surface coat molecules are present on all areas of the ciliary membrane of rat tracheal epithelia.     

Ultrastructural localization of anionic sites on the surface of yeast, hyphal and germ-tube forming cells of Candida albicans

The cell wall of Candida albicans contains chitin, beta-glucans and phosphorylated mannoproteins, and possesses a fuzzy coat which is thought to play a role in pathogenicity, phagocytosis, and adherence of this dimorphic yeast. Using scanning electron microscopy and the gold method, mannoproteins were detected on the whole surface of blastoconidia including the bud scars, but chitin was absent even after alpha-mannosidase treatment of the cells. The presence of surface beta-(1----6)glucan (but not beta(1----3)glucan) was observed only after extensive alpha-mannosidase and alkaline phosphatase treatments of blastoconidia. Using transmission and scanning electron microscopy, the locations of anionic sites were revealed by polycationic colloidal gold-chitosan complexes on the surface of blastoconidia, germ tubes and hyphae. Anionic sites were dispersed evenly over the surface of blastoconidia bearing bud scars. Depending upon the growth conditions, anionic sites could be detected on emerging buds and young cells. However, bud scars were always free of marking. When germ-tube formation was induced, anionic sites were present at different densities on all cell surfaces, the highest density being observed on cells with bud scars. Anionic sites were detected at a remarkably high density on all hyphal surfaces. An apical concentration of anionic sites was observed on germ tubes and hyphae. The distribution of anionic sites was not modified by endoglucosaminidase treatment of blastoconidia, germ tubes and hyphae. The anionic sites were associated with the fuzzy coat. As the hyphal form is regarded as possessing the greatest invasiveness, it is suggested that anionic sites play an important role in establishing tissue colonization by this human pathogen.