Topographical differences in the distribution of surface coat components and intramembrane particles. A cytochemical and freeze- fracture study in culture forms of Trypanosoma cruzi

A regional specialization of the cell surface of T. cruzi culture forms was found at the cytostome as a localized thick surface coat rich in carbohydrate-containing components. The prominent surface coat was located over a region of the plasma membrane where intramembranous particles were exceedingly low in number. In turn, the particle-poor region was related to specialized submembrane fibrils not present under other regions of the plasma membrane. The cystostome region provides a striking example of a stable regional differentiation of the plasma membrane, involving the outer surface, the membrane interior, and the underlying cytoplasm. In addition, independence of Con A receptors, colloidal iron binding sites, and ruthenium red-stainable surface components from membrane particles was demonstrated at the flagellar membrane.     

Electron microscopic observations of the surface of L-cells in culture

Summary Techniques are described for the preparation of preshadowed replicas of both the upper and lower surfaces of L-cells in culture, and of cross sections of L-cells growing on a cellophane substrate. These revealed long slender microvilli, 800 to 1,100 A in diameter, projecting from both upper and lower surfaces of the cells. These microvilli were frequently observed to contact other cells and substrate, and to leave material behind on the substrate. The plasma membrane of the lower surface was separated from the substrate by an electron-lucent gap 200 to 300 A wide. The surface coat of the L-cell was visualized by staining with colloidal iron and ruthenium. Staining with colloidal iron was most intense on the surface of the microvilli. The gap between cell and substrate was intensely stained with ruthenium red. Enzymatic digestion of living cells revealed that both trypsin and neuraminidase reduced the staining of the cell coat by colloidal iron, whereas only trypsin altered its staining with ruthenium red. After trypsin treatment, fragments of an amorphous material with the staining characteristics of the cell coat were observed between the denuded cells. Treatment with ribonuclease, chymotrypsin or hyaluronidase did not affect the staining of the cell coat.     

Ultrastructural localization of concanavalin A receptors in the plasma membrane: association with underlying actin filaments

Whole-mount cell preparations of cultured rat 3Y1 cells were examined by stereo electron microscopy to identify the ultrastructural localization of concanavalin A (Con A) receptors in the plasma membrane, and to clarify the relationship between Con A receptors and cytoskeletal components. Well spread monolayer cells were extracted with saponin, briefly fixed, and then partially broken open with shearing force to facilitate the introduction of antibodies for identification of actin filaments. Stereo electron microscopy of such treated cells revealed a 3-dimensional image of filamentous structures such as fine filaments, microtubules (MT) and endoplasmic reticulum (ER) in the flattened areas of each cell. Just beneath the plasma membrane were meshworks of actin-containing fine filaments, as identified by an immunogold staining method. Microtubules and ER were observed to be either directly or indirectly associated with this meshwork. The broken open part of each cell exhibited a meshwork of filaments which were associated with the cytoplasmic surface of the plasma membrane. Some of the filaments were connected to the plasma membrane either by their ends or by their lateral surfaces. The localization of Con A receptors was examined by binding colloidal gold-labelled Con A to the surface of fixed, saponin-extracted cells. Virtually all gold particles bound externally at the same membrane sites where intracellular actin filaments attached internally. The observations strongly suggest that the distribution of Con A receptors was regulated by the underlying meshwork of actin filaments.     

Ultracytochemical Modifications of Surface Carbohydrates in Fertilized Eggs of the Common Carp

The surface of the plasma membrane of unfertilized and fertilized carp eggs was examined by four cytochemical techniques, colloidal thorium, colloidal iron, ruthenium red and phosphotungstic acid stainings, to determine the carbohydrate moieties. The surface of the plasma membrane of unfertilized eggs stained only with colloidal iron, which was heterogeneously deposited: no deposits were seen on the plasma membrane near overlying cortical alveoli. In fertilized eggs, the membrane was stained by all four methods. These ultracytochemical modifications of the surface of the plasma membrane may be caused by participation of the limiting membranes of secretory organelles, probably by turnover of the inner surface of the limiting membranes. Neuraminidase treatment of fertilized eggs eliminated the deposits of colloidal iron on the surface of the plasma membrane and caused an increase in stainability with ruthenium red. Treatment with neuraminidase or trypsin prevented the staining with phosphotungstic acid.     

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.)

Summary Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.For all correspondence     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

An electron microscopic investigation of the surface coat of the electrocyte of Electrophorus electricus

Summary The surface coat of the electrocyte of the main electric organ of Electrophorus electricus was studied using cytochemical methods (periodic acid-silver methenamine, periodic acid-chromic acid-silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, Concanavalin A — horseradish peroxidase, ruthenium red, Alcian-blue lanthanum nitrate, colloidal iron hydroxide and cationized ferritin). The surface of the electrocyte presents perpendicularly oriented tubular invaginations of the cell membrane. The fibrous coat 50–100 nm thick, penetrates into the lumen of the invaginations. It is also observed in the synaptic clefts existent in the posterior face of the electrocyte. The coating of the surface membrane gives a positive reaction with all techniques used. Binding of colloidal iron hydroxide particles was observed only in the outer layer of the coat. With the Alcian-blue lanthanum nitrate technique, microtubules were observed in the cytoplasm of the electrocyte.The results indicate that the surface coat of the electrocyte contains mucopolysaccharides, glycoproteins, acid mucopolysaccharides and anionic sites detected at low (colloidal iron hydroxyde) and neutral (cationized ferritin) pH.This work has been supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Conselho de Ensino e Pesquisa da UFRJ (CEPG) and Banco Nacional de Desenvolvimento Econômico     

Alterations in intramembrane particle distribution during interaction of erythrocyte-bound ligands with immunoprotein receptors

Freeze fracture studies have been performed on rabbit pulmonary alveolar macrophages and a nonphagocytic murine lymphoblastoid cell line, PU-5 Fc+, incubated with sheep erythrocytes, sheep erythrocyte-IgG Forssman antibody complex, sheep erythrocyte-IgG Forssman antibody-C complexes and aggregated IgG. Alveolar macrophages show redistribution of intramembrane particles after interaction with (EIgG) and E(IgM)C. The murine lymphoblastoid cell line shows intramembrane particle redistribution consequential to binding of E(IgG) and aggregated IgG. The results demonstrate that after specific immunoprotein receptor-ligand interaction, there is extensive plasma membrane reorganization which results in a redistribution and loss of intramembrane particles. Changes are observed in the protoplasmic face of the plasma membrane after the binding of ligand to the outer membrane surface. The findings suggest that interaction of erthrocyte-bound ligands with specific lymphoid and macrophage plasma membrane receptors leads to a generalized redistribution of integral membrane components in the membrane.     

Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells

Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2–3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10^?7–10^?5 M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.     

Reversibility of cell surface label rearrangement

Cell surface labeling can cause rearrangements of randomly distributed membrane components. Removal of the label bound to the cell surface allows the membrane components to return to their original random distribution, demonstrating that label is necessary to maintain as well as to induce rearrangements. With scanning electron microscopy, the rearrangement of concanavalin A (con A) and ricin binding sites on LA-9 cells has been followed by means of hemocyanin, a visual label. The removal of con A from its binding sites at the cell surface with alpha-methyl mannoside, and the return of these sites to their original distribution are also followed in this manner. There are labeling differences with con A and ricin. Under some conditions, however, the same rearrangements are seen with both lectins. The disappearance of labeled sites from areas of ruffling activity is a major feature of the rearrangements seen. Both this ruffling activity and the rearrangement of label are sensitive to cytochalasin B, and ruffling activity, perhaps along with other cytochalasin-sensitive structure, may play a role in the rearrangements of labeled sites.     

Cell surface changes on the mouse blastocyst at implantation

Cell surface changes on the trophectoderm of the mouse blastocyst have been followed in the periimplantation period using electronhistochemical techniques. Examination of the ability of the trophectoderm to bind positively charged colloidal iron particles before and after enzyme treatment has shown that sialic acid-containing glycoproteins make a considerable contribution to the negative charge on the blastocyst surface. At implantation these membrane components are lost or undergo modification independently of direct maternal influence as indicated by a marked decline in colloidal iron binding at this time, both in vivo and in vitro. The findings are discussed in relation to other surface changes on the blastocyst and to the initiation of implantation.     

Modulation of cell surface iron transferrin receptors by cellular density and state of activation

This report describes investigations of plasma membrane transferrin receptors on a variety of lymphoid cell lines and normal peripheral blood lymphocytes during activation and cell growth cycles. Transformed lymphoid cell lines have as many as 1,000 times the number of receptors found on normal resting lymphocytes. The number of iron transferrin receptors on continuous cell lines as well as normal human fibroblasts is down-regulated during the transition from log-phase growth to stationary plateau growth. When normal lymphocytes are transformed by mixed lymphocyte culture or mitogens, they rapidly express a 50-fold increase in the number of transferrin binding sites. This appearance of iron transferrin receptors anticipates nuclear changes during cell activation and subsequent mitosis of normal cells.     

Restriction of a sperm surface antigen's mobility during capacitation.

Fluorescent Fab¹ [immunoglobulin G (IgG) fragment with antigen binding capacity from papain digestion of IgG] antibody fragments and globulin from antisera prepared against a single rabbit sperm surface membrane glycoprotein antigen (MGP) were used to study surface antigen mobility. Epididymal and ejaculated spermatozoa exhibited a redistribution of MGP surface antigen over the acrosomal region when labeled at 4°C and warmed to 37°C. Following in vivo capacitation, spermatozoa did not exhibit MGP surface antigen redistribution over the acrosome. The restricted mobility of this surface antigen implies a physiological change in plasma membrane fluidity, which may be a necessary preliminary to the acrosome reaction. Furthermore, it is suggested that the presence or absence of specific peripheral membrane proteins may control the positional relationship between mobile and nonmobile integral membrane components.     

Recognition of Entamoeba histolytica 115-kDa surface protein by human secretory immunoglobulin A antibodies from asymptomatic carriers

Entamoeba histolytica is a protozoan parasite that can invade the intestinal mucosa. Infection induces production of secretory immunoglobulin A (SIgA) antibodies that can diminish the adhesion between E. histolytica trophozoites and epithelial cells in vitro and reduce the rate of new infections in children. SIgA antibodies produced by asymptomatic cyst carriers could play a protective role against the damage caused by E. histolytica. To identify membrane antigens capable of inducing SIgA response in E. histolytica cyst carriers, salivary SIgA antibodies were confronted with blotted plasma membrane proteins from amebae. A surface 115-kDa ameba protein was recognized by 62% of the human SIgA antibodies tested. The 115-kDa protein is not a mannose-containing glycoprotein and has no protease activity. Rabbit anti-115-kDa protein antibodies were capable of reducing erythrophagocytosis but were unable to protect culture cells from the cytopathic damage caused by E. histolytica. However, anti-115-kDa protein antibodies induced surface receptor redistribution.     

Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

Colloidal alpha-stannic acid and negative iron colloid as differential electron stains for surface proteins.

Colloidal alpha-stannic acid and a negative iron colloid obtained from ferric hydroxide and potassium ferrocyanide, both negative sols being stable within a wide pH range, were refined as surface protein electron markers. Because of the relatively small size of its particles, colloidal alpha-stannic acid was used for staining all surface proteins. According to the pH at which the negative iron colloid was applied, it revealed either all surface proteins, or because of its large colloidal particles, stained basic proteins. This differential staining capability of the iron colloidal has been demonstrated previously on various control preparations (Puvion E, Blanquet PR: J Microsc 12:171, 1971). Controls on the affinity of the two colloids to surface amino groups were carried out on rat liver, mouse fibroblasts, HeLa and KB cells, Ehrlich and Zajdela ascites cells subjected to prior enzymatic and chemical treatments (incubation with neuraminidase or phospholipase C, esterification, acetylation or lipid extraction). At any pH below 9, the two sols stained proteins in the outer hydrophilic leaflet of esterified cells with relative selectivity, but the alpha-stannic acid showed them more accurately. The iron sol did reveal at high pH protein components of high isoionic point on the surfaces of rat hepatocytes and ascites cells which had only been treated with neuraminidase.     

Interaction of Concanavalin A and a Divalent Derivative with Lymphocytes and Reconstituted Lymphocyte Membrane Glycoproteins

Both concanavalin A (con A) and its divalent derivative, succinyl-concanavalin A (S-con A) are mitogenic for porcine lymph node lymphocytes. We have compared the binding of these two lectins to intact porcine lymphocytes and phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins. Both con A and S-con A showed high- and low-affinity binding to intact cells, as indicated by LIGAND analysis of Scatchard plots of binding data. Despite the apparently identical saccharide specificities of the two lectins, high-affinity binding sites for S-con A were only one-third as numerous as high-affinity sites for the parent lectin. Large numbers of low-affinity binding sites existed for con A, while many fewer were present for S-con A. It is suggested that these sites result from hydrophobic association. Con A bound to lymphocytes in a positively cooperative fashion, while S-con A showed non-cooperative behavior. Lectin binding to large unilamellar phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins was measured using a rapid filtration assay, and was linear with the glycoprotein content of the vesicles. Almost all of the outward-facing glycoprotein was functional in terms of lectin binding. Reconstituted glycoproteins showed only a single class of high-affinity binding sites for both con A and S-con A, with association constants similar to those measured for intact cells. Con A, but not S-con A, showed positively cooperative binding to reconstituted vesicles. Cooperativity was observed in both gel phase and liquid crystalline phase lipid, and was thus not dependent on long-range lateral rearrangement of glycoprotein receptors. Results suggested that con A induces a microre-distribution of receptors on the lymphocyte membrane surface, leading to the exposure of glycoproteins that were previously inaccessible to the lectin. S-Con A does not cause glycoprotein redistribution, and a large fraction of the receptors remain cryptic.     

Interaction of concanavalin A and a divalent derivative with lymphocytes and reconstituted lymphocyte membrane glycoproteins

Both concanavalin A (con A) and its divalent derivative, succinyl-concanavalin A (S-con A) are mitogenic for porcine lymph node lymphocytes. We have compared the binding of these two lectins to intact porcine lymphocytes and phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins. Both con A and S-con A showed high- and low-affinity binding to intact cells, as indicated by LIGAND analysis of Scatchard plots of binding data. Despite the apparently identical saccharide specificities of the two lectins, high-affinity binding sites for S-con A were only one-third as numerous as high-affinity sites for the parent lectin. Large numbers of low-affinity binding sites existed for con A, while many fewer were present for S-con A. It is suggested that these sites result from hydrophobic association. Con A bound to lymphocytes in a positively cooperative fashion, while S-con A showed noncooperative behavior. Lectin binding to large unilamellar phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins was measured using a rapid filtration assay, and was linear with the glycoprotein content of the vesicles. Almost all of the outward-facing glycoprotein was functional in terms of lectin binding. Reconstituted glycoproteins showed only a single class of high-affinity binding sites for both con A and S-con A, with association constants similar to those measured for intact cells. Con A, but not S-con A, showed positively cooperative binding to reconstituted vesicles. Cooperativity was observed in both gel phase and liquid crystalline phase lipid, and was thus not dependent on long-range lateral rearrangement of glycoprotein receptors. Results suggested that con A induces a microredistribution of receptors on the lymphocyte membrane surface, leading to the exposure of glycoproteins that were previously inaccessible to the lectin. S-Con A does not cause glycoprotein redistribution, and a large fraction of the receptors remain cryptic.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules.

The distribution of concanavalin A (con A) receptor sites on the membranes of chromaffin granules has been investigated by binding studies using 125I-labelled con A and by electron-microscope studies using ferritin-labelled con A. In both experiments con A was observed to bind to chromaffin granule membranes but not to intact granules. The ferritin-con A particles bind to only one of the two possible surfaces of the chromaffin granule membranes. These results are in agreement with previous observations concerning the asymmetric distribution of saccharide residues on the surfaces of a number of different plasma membranes. They suggest that for the intracellular membrane of the chromaffin granule the saccharide sites, like those in plasma membranes, are not exposed to the cell cytoplasm. Further work is necessary to establish whether these sites are on the inner surface of the membrane or whether they are unmasked during the conversion of granules to membrane ghosts.