Sialic acid is a cell surface component of Entamoeba invadens trophozoites

The surface anionic groups of Entamoeba invadens were analysed by cell electrophoresis, by ultrastructural cytochemistry, and by identification of sialic acids using paper and gas-liquid chromatography. Binding of colloidal iron hydroxide (CIH) and of cationized ferritin (CF) particles at pH 1.8 and 7.2, respectively, was observed on the cell surface. E. invadens has a highly negative surface charge (-0.96 microns s-1 V-1 cm). Treatment of the cells with trypsin and neuraminidase significantly reduced the electrophoretic mobility by 24% and 40%, respectively. Treatment of the amoebae with neuraminidase also markedly decreased the binding of CIH to the cell surface. This finding suggests that sialic acid residues are the major anionogenic groups exposed on the surface of E. invadens. Paper and gas-liquid chromatography showed that N-acetylneuraminic acid was the only derivative characterized in E. invadens.     

Radiation-induced changes of negative charge on the cell surface of primary human fibroblasts

By binding cationized ferritin (CF) to the plasma membrane of primary human fibroblasts, the amount and topology of negatively charged sites on cell surfaces were studied after X-irradiation. The CF binding was tested both on fixed and unfixed cells. Using various enzymes, the chemical nature of sites carrying the negative charges on cell surfaces was investigated. The results suggest that in unirradiated fibroblasts the CF binding occurred in a polarized manner, i.e. the particles were localized mainly on the apical surface of cells and formed clusters. The thin cytoplasmic protrusions and cell-to-cell contact sites bound CF to a greater extent than the bleb-like formations. Enzymatic digestion of surface polysaccharides showed that the main carriers of negatively charged sites are the glycosaminoglycans associated with the cell surface. The fixation of cells with glutaraldehyde did not influence the topology of CF binding either before or after the enzymatic treatment. After X-irradiation with 2.5 Gy the topology of CF binding did not change but the CF coverage of cells as well as the amount of ferritin particles per unit of surface area decreased within 10 min. The changes proved to be reversible as the values reached the pre-irradiation level by 1 h after irradiation.     

Binding of cationized ferritin to the cell-coat glycoproteins of human and rat small-intestinal absorptive cells

Summary The binding of cationized ferritin (CF) to the cell-coat (glycocalyx) glycoproteins of human and rat intestinal absorptive cells was investigated in relation to the amount of sialic acid in these macromolecules. The cell coat of human absorptive cells exhibited poor binding of CF and contained a small amount of sialic acid. The cell coat of rat absorptive cells had about ten times more sialic acid than that of human cells and showed a strong affinity for the marker. The removal of sialic acid from the cell-coat glycoproteins of rat intestinal cells by neuraminidase treatment abolished CF binding. These results suggest that sialic acid is necessary for CF binding and that human and rat intestinal absorptive cells show a species-specific difference in the sugar composition of the cell coat.     

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.     

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.     

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.     

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.     

Chemical determination of cationized ferritin bound to synaptosomes

Surface charge of synaptosomes was studied with cationized ferritin (CF) binding at pH between 4 and 7. The characteristic of binding resembles that of the electrophoretic mobility and suggests an electrostatic interaction between CF and membranes. When sialic acid was partially removed from synaptosomes, a reduction in binding was detected. This suggests that sialic acid is one determinant of surface charge of synaptosomes. The present method differs from other CF studies in that the amount of bound CF was measured chemically instead of by electron microscopy. This method could be useful in other cell systems.     

Endocytosis by platelets during cationized ferritin-induced aggregation

Summary Localization of cationized ferritin (CF) particles in the process of CF-induced aggregation of rabbit platelets was investigated by electron microscopy. CF particles attached to the surface membrane of discoidal platelets immediately after the addition of CF. Some platelets were connected to each other through the CF particles located on their surfaces. At 30 s after the addition of CF, aggregation of platelets in round form was observed. During the time course of aggregation, CF particles moved to the interplatelet spaces. Also CF particles were found in the open canalicular system, the membrane component of which was stained with ruthenium red. On the other hand, CF particles were also found in ruthenium-red-negative vesicles in platelets. At 180 s after, CF particles containing vacuoles, which showed acid phosphatase activity, were observed in the aggregates. These results suggest that part of CF particles may be incorporated into the cytoplasma by endocytosis.     

Freeze-fracture cytochemistry of rat glomerular capillary tuft. Determination of wheat germ agglutinin binding sites and localization of anionic charges

We propose here the use of freeze-fracture to gain access and to label in vitro glomerular components and locate WGA receptors and anionic sites. Tissues are frozen, fractured under liquid nitrogen, and thawed. Freeze-fracture rendered all glomerular structures directly accessible to the reagents. This made possible study of the nature and topology of cationized ferritin and WGA binding sites. WGA-gold complexes were observed over plasma membranes of podocytes and of endothelial and mesangial cells. Labeling of podocytes and endothelial cells was similar in the mesangial area and in the peripheral part of the capillary loop. Cross-fractures of extracellular matrices showed that WGA bound uniformly to the glomerular basement membrane (GBM) as well as to mesangial matrix. In fractured specimens treated with neuraminidase, WGA was no longer observed over podocytes but it consistently labeled the surface of endothelial and mesangial cells. Whereas in GBM cross-sections WGA binding was greatly reduced or even abolished, it remained unmodified in the mesangium. This shows that only NeuNAc (sialic acid) might account for the binding of WGA to podocytes, whereas GlcNAcs appear to be the main WGA binding sites on endothelial and mesangial cells and in the mesangial matrix. Both NeuNAc and GLcNAc residues are probably associated in GBM. With cationized ferritin (pI 8.3) at pH 7.4, intense, continuous labeling was seen all over the different plasma membranes, denser in podocytes than in endothelial cells. CF was also observed in cross-fractured profiles of extracellular matrices and never appeared agglutinated in discrete sites.     

Colloidal iron hydroxide-binding to the surfaces of chick embryo fibroblasts transformed by wild-type and a temperature-sensitive mutant of Rous sarcoma virus.

The densities of colloidal iron hydroxide (CIH) particles binding to the surfaces of chick embryo fibroblasts were determined before and after transformation with wild type Rous sarcoma virus and a temperature sensitive (ts) mutant of this virus. On the basis of in vitro behavior, cells transformed by the ts virus manifest a malignant phenotype at 36 degrees C (permissive temperature) and appear normal at 41 degrees C (non-permissive temperature). At the permissive temperatures there is a significant increase in CIH particle-binding to spaces of cell surface between microvilli on the wild type and ts transformed cells. At the non-permissive temperature this significant increase in binding is only observed on the wild type transformant, while the density found on the ts transformant is not significantly different from the untransformed state. Therefore, in vitro characteristics of normalcy and malignancy are reflected in changes in the CIH binding properties of the cell surface spaces between microvilli. The CIH densities observed on the microvilli are significantly different from the density on the spaces between them for each of the classes of cells studied at either temperature. The microvilli are found to bind a lower density of particles in five of the six cases. No correlations between microvilli particle density and transformation to in vitro malignant characteristics were observed.     

Endocytosis in guinea pig seminal vesicle epithelial cells cultivated in chemically defined medium

We have developed a chemically defined monolayer culture system for guinea pig seminal vesicle epithelial cells (SVEP). The cells appeared as a polarized monolayer with apical microvilli, tight junctions and desmosome-like junctions, and often dilated intercellular spaces. SVEP expressed epithelial-specific cytokeratins as detected immunocytochemically. Growth was obtained during the first week of culture. In this period, the cells were exposed to unconjugated horseradish peroxidase (HRP), a ricin-peroxidase conjugate (Ri-HRP), or cationized ferritin (CF). HRP was endocytosed without binding to the SVEP surface (fluid-phase endocytosis) and was found mainly in multivesicular endosomes and lysosomes. Ri-HRP and CF, however, were endocytosed following binding to the cell surface. Initially these markers were present in multivesicular endosomes, but later also in smaller tubular and vesicular endosomes, some Golgi-associated elements (but not Golgi stacks), and lysosomes. We conclude that our SVEP culture system may be useful in further studies, on e.g. hormonal regulation of endocytosis and other processes of importance for SVEP maintenance and modulation of the seminal fluid in vivo.     

The Surface Charge of Tritrichomonas foetus

The surface charge of Tritrichomonas foetus was evaluated by means of the binding of colloidal iron hydroxide particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM), of cells suspended in solutions of different ionic strength and pH. At pH 7.2, T. foetus has a negative surface charge with a mean EPM of ?1.03 μmμs^?1μV^?1μcm. At lower pH, there is a decrease in the negative surface charge with an isoelectric point at pH 1.2. At higher pH (> 9.0), there is an increase in the surface charge reaching an EPM of ?2.5 μmμs^?1μV^?1μcm. These results indicate that the surface of T. foetus contains both negatively and positively charged dissociating groups. Binding of colloidal iron hydroxide and cationized ferritin particles throughout the cell surface of the protozoon was observed. Treatment of T. foetus with neuraminidase or trypsin reduced significantly the EPM of the cells. Enzyme-treated cells recovered their normal EPM when incubated for 6 h in fresh culture medium by a process that is inhibited by puromycin.     

Cationized ferritin as a platelet-stimulating surface probe. Binding to platelets and effects on platelet function

Polycationic derivatives of ferritin containing primary amino groups (CFah) or tertiary amino groups (CFdmp) were potent platelet agonists inducing shape change, aggregation and secretion, but also agglutination in the presence of EDTA. Pretreatment of platelets with neuraminidase, PGE1, indomethacin, or creatine kinase/creatine phosphate inhibited CF-induced activation. In contrast, neuraminidase and PGE1 increased the agglutination by CF, indicating an inverse relationship between activation and CF-induced agglutination. At pH 7.4, the cationic charges of CFdmp exceeded those of CFah by a factor of 1.5 and the platelets bound approximately 1.5 times more CFah than CFdmp, suggesting the same number of anionic surface sites for both CF preparations. The capacity of the platelets to bind CF was diminished by 55% at 0 degree C or by 62% after aldehyde fixation and by 13% with PGE1. This suggests that the binding capacity depends on the mobility of the binding sites in the plane of the membrane but is only slightly increased by platelet activation. Binding to fixed or cold platelets approached equilibrium within a few seconds whereas saturation required several minutes at 37 degrees C. Neuraminidase preferentially reduced the slow binding and much less the rapid binding. Since activation by CF developed during seconds, suppressible by a brief treatment with neuraminidase 25 mU/ml, a small portion of neuraminidase-sensitive sites appears to be necessary for CF-induced platelet activation. Full activation and agglutination occurred at CF concentrations far below saturating concentrations. The results show that neither CF-induced activation nor agglutination depend on a simple neutralization of the negative surface charge.     

Distribution of electric charges and concanavalin A binding sites on autophagic vacuoles and lysosomes in mouse hepatocytes

The distributions of electric charges and Concanavalin A binding sites in autophagic vacuoles and lysosomes in mouse hepatocytes were studied by utilizing a frozen ultrathin section labeling method with cationized ferritin (CF) or anionized ferritin and ferritin-conjugated Concanavalin A (Con A-F) as visual probes. Our observations revealed that the inner surface of the autophagic vacuole membrane has more anionic sites (CF binding) than other organelle membranes. This suggests that if the limiting membranes of autophagic vacuoles originate from preexisting membranes, such membranes must undergo structural and compositional alternation during the formation of the autophagic vacuoles. In contrast to CF, Con A-F showed no distinct binding to the membranes of autophagic vacuoles, but the contents of vacuoles displayed varying Con A-F binding, depending on the stage of the autophagic process. Increased binding was seen in more mature autophagic vacuoles. Since lysosomes showed a preferential accumulation of Con A-F particles, molecules with Con A-F binding sites in autophagic vacuoles may be of lysosomal origin. Con A-F distribution varied from lysosome to lysosome in the same cell, indicating heterogeneity of lysosomal contents. These results suggest that ferritin-conjugated lectin labeling methods applied to frozen, ultrathin section are a useful new approach in analyzing the natural history of autophagic vacuoles and the heterogeneity of lysosomes.     

Vesicular transport of cationized ferritin by the epithelium of the rat choroid plexus

We have studied the transport of ferritin that was internalized by coated micropinocytic vesicles at the apical surface of the choroid plexus epithelium in situ. After ventriculocisternal perfusion of native ferritin (NF) or cationized ferritin (CF), three routes followed by the tracers are revealed: (a) to lysosomes, (b) to cisternal compartments, and (c) to the basolateral cell surface. (a) NF is micropinocytosed to a very limited degree and appears in a few lysosomal elements whereas CF is taken up in large amounts and can be followed, via endocytic vacuoles and light multivesicular bodies, to dark multivesicular bodies and dense bodies. (b) Occasionally, CF particles are found in cisterns that may represent GERL or trans-Golgi elements, whereas stacked Golgi cisterns never contain CF. (c) Transepithelial vesicular transport of CF is distinctly revealed. The intercellular spaces of the epithelium, below the apical tight junctions, contain numerous clusters of CF particles, often associated with surface-connected, coated vesicles. Vesicles in the process of exocytosis of CF are also present at the basal epithelial surface, whereas connective tissue elements below the epithelium are unlabeled. Our conclusion is that fluid and solutes removed from the cerebrospinal fluid by endocytosis either become sequestered in the lysosomal apparatus of the choroidal epithelium or are transported to the basolateral surface. However, our results do not indicate any significant recycling via Golgi complexes of internalized apical cell membrane.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.     

Cell membrane polarity in primary human fibroblasts

The topologies of bindings of cationized CF and native ferritin (NF) on plasma membranes of primary human fibroblasts were examined by transmission electronmicroscopy. Both ligands bound mainly to apical surfaces and less abundantly to lateral regions. At basal localization they were observed only at cell-to-cell connections. The polarity of negatively charged binding sites was not altered by glutaraldehyde fixation. The definite polarity of plasma membranes of cultured cells express alternating surface areas charged negatively or positively.     

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

Membrane retrieval in epithelial cells of isolated thyroid follicles.

Follicles from rat and pig thyroid glands were isolated by digestion with collagenase. The epithelial cells of isolated follicles maintain their structural and functional polarity as shown by incorporation of 3H-leucine and autoradiography. To trace the fate of surface membrane, isolated follicles were opened, stimulated with thyrotropin and incubated for various time intervals with cationized ferritin (CF), uncharged dextran, native ferritin (NF), and latex spheres (0.5 mum in diameter) which were either pre-coated with CF or added together with CF. Uncharged dextran and native ferritin did not bind to the luminal cell membrane, were taken up in small amounts and accumulated in lysosomes; anionic NF was not found in Golgi cisternae in contrast to uncharged dextran which occassionally reached a few Golgi stacks. CF bound rapidly and in clusters to the luminal plasmalemma, preferentially to coated pits, was taken up by endocytosis, accumulated in lysosomes after 5 min and reached the Golgi cisternae after 30 min. Latex spheres were taken up by engulfment through fusion of microvilli and reached the lysosomes. CF particles coating the latex spheres may detach at this station and reach the Golgi cisternae. The findings show that the route of small tracers depends on the charge of the tracer, in agreement with results obtained by Farquhar [8]. Vesicles carrying NF can be traced to lysosomes only, whereas vesicles containing uncharged dextran or - more conspicuously -CF also fuse with Golgi membranes. Large tracers (latex beads) reach only the lysosomes, but CF taken up with them may move to Golgi cisternae.