Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

Detection of biospecific interactions using amplified ellipsometry

Amplified detection of biomolecules and biological interactions using an optical surface technique, ellipsometry, is demonstrated for two biosystems--immunoglobulin G with anti-immunoglobulin G (IgG) and the lectin concanavalin A (Con A) with yeast cells. In order to improve the sensitivity of the ellipsometer signal, an amplifier conjugate is formed by binding the affinity ligand to a 12-nm silica particle which is readily detected by the ellipsometer. Thus by using conjugates of IgG-silica and Con A-silica, amplifications of five to seven times have been obtained enabling detection of less than 20 pg/mm2 of biomolecular material.     

Lectin-bearing liposomes: differential binding to normal and to transformed mouse fibroblasts

The binding of covalent conjugates of concanavalin A (Con A) or wheat germ agglutinin (WGA) and liposomes (lectin-liposomes) to the surface of normal and transformed mouse fibroblasts was studied. Quantitation of the binding was performed by means of microfluorometry and radioactive lipid label counting using both sparse and dense cell cultures. It was found that 2.5-3 times more lectin-conjugated liposomes are bound to L or SV3T3 cells than to the mouse embryo fibroblasts and 3T3 cells in a broad concentration range. The binding of Con A- and WGA-liposomes was inhibited up to 70% in the presence of the corresponding carbohydrate inhibitors. A decreased binding of lectin-liposomes to cells was also observed when cells were pretreated with the free lectin. Trypsinization of the cells resulted in an increase in the Con A-liposomes binding to normal fibroblasts. When free fluorescent Con A or WGA was used in binding studies no profound differences in the binding of lectin to normal or transformed cells were detected. The relation of the lectin-liposome/cell to cell/cell interactions is discussed.     

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes.     

Cytochemical analysis of oligosaccharide processing in frog photoreceptors

Summary Lectin cytochemistry, together with exoglycosidase enzyme digestion, has been used to characterize partially glycoconjugates of several intracellular compartments in frog photoreceptors. In order to obtain uniform access of reagents to all intracellular compartments, the experiments were performed directly on semi-thin sections ofXenopus laevis retinal tissue embedded in a hydrophilic plastic resin. In the rod, the major photoreceptor intracellular binding sites for wheat germ agglutinin (WGA) are the outer segment, the Golgi complex, and other inner segment organelles which are probably involved in the transport of glycoconjugates from the Golgi complex to the outer segment. In addition, shed outer segment tips (phagosomes) are uniformly labelled with WGA. The WGA-binding sites of the outer segment and of the presumed transport organelles are resistant to neuraminidase digestion. This is consistent with the possibility that glycoconjugates (primarily opsin) are transported from the Golgi complex to the outer segment without further oligosaccharide processing. Specific staining of rod outer segments and of phagosomes is also obtained with theN-acetylglucosamine-specific lectin, succinyl-WGA (S-WGA). Outer segments and phagosomes stain the same with WGA, S-WGA and a variety of other lectins tested suggesting that no major post-Golgi oligosaccharide processing accompanies the shedding-phagocytosis event. Concanavalin A (Con A) staining of intracellular sites in rod inner segments reveals a striking difference compared to WGA staining in that the Con A binding sites are concentrated in the photoreceptor axon and presynaptic terminal. These results, and results from previous studies, indicate that the photoreceptor may utilize different mechanisms of oligosaccharide processing from the level of a single Golgi complex to the opposite ends of this cell. Furthermore, those glycoconjugates destined for the presynaptic terminal may undergo post-Golgi processing at or near their sites of insertion into the presynaptic plasma membrane.     

Concanavalin A receptors, immunoglobulins, and theta antigen of the lymphocyte surface. Interactions with concanavalin A and with Cytoplasmic structures

The effect of concanavalin A (Con A) on the capping of mouse lymphocyte surface immunoglobulin (surface Ig), cross-linked by rabbit anti-mouse Ig antibody, and on the capping of mouse thymocyte theta antigen, cross- linked by anti-theta alloantibody and rabbit anti-mouse Ig antibody, has been studied by immunofluorescence, using fluorescein conjugated Con A and rhodamine-conjugated anti-mouse Ig antibody, and by electron microscopy, using native or fluorescein-conjugated Con A and ferritin- conjugated anti-mouse Ig antibody. Prior incubation of the cells with Con A inhibited only partially capping os surface Ig, whereas it blocked almost completely capping of theta antigens. Both on cells with rings and on cells with caps the staining for surface Ig or theta antigen was superimposed to the staining for Con A. When Con A receptors on spleen cells were capped by Con A at concentrations of 10 mug/ml or higher, and the distribution of surface Ig was examined under noncapping conditions, all detectable surface Ig were found in the caps. As shown by electron microscopy, surface Ig remained dispersed in a layer of Con A. The ability of Con A to cap surface Ig was not altered by the presence of cohchicine or vinblastine. These results suggest that surface Ig are cross-linked by Con A to other Con A receptors. In these conditions surface Ig behave essentially as Con A receptors, as for example, in their sensitivity to cytochalasin B during inhibition or reversal of capping induced by this drug. The behavior of surface Ig parallels that of Con A receptors also in the presence of vinblastine. It is concluded that in the presence of Con A, antimitotic drugs do not modify directly the interaction between Con A receptors and surface Ig, but probably influence the capping ability of the Con A receptors or, more in general, affect the ability to elicit movements over the cell surface. The role in capping of cytochalasin- sensitive and vinblastine-sensitive structures is discussed. Both types of structures appear to play an active role in the formation of a cap, although the former probably corresponds to the main mechanical system responsible for the active displacement of cytoplasmic and surface material.     

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.     

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.     

Does concanavalin A treatment of host cells enhance or inhibit their association with Trypanosoma cruzi trypomastigotes?

Concanavalin A (Con A) has frequently been used as a probe of cell surface molecules that mediate cell-cell interactions. There have been conflicting reports that Con A treatment of vertebrate host cells can subsequently increase or reduce the level of association (surface attachment and penetration) of Trypanosoma cruzi trypomastigotes with these cells. In this work, we have established that the type of effect produced by treatment of host cells with Con A depended on whether or not fetal bovine serum was present during the interaction of trypomastigotes and host cells; Con A treatment reduced host cell association with T. cruzi in the presence of the serum, but increased it when the serum was absent. In addition, ovalbumin, a glycoprotein with a high mannose content and the ability to specifically bind to Con A, was found capable of altering the effect of Con A treatment of host cells on parasite association levels in a manner similar to fetal bovine serum. These results suggested that glycoproteins present in the serum can modulate the effect of Con A, possibly by blocking free sites remaining on the Con A molecules which had bound to the surface of host cells. If free binding sites on the Con A molecule remained unblocked, they could conceivably form bridges between host cells and parasites resulting in an artifactual enhancement of their level of association in serum-free medium.     

Distribution and mobility of concanavalin a receptors on isolated guinea pig epidermal cells at various stages of differentiation

Trypsinized guinea pig epidermal cells were separated by velocity sedimentation at unit gravity. Based on the relationship between cell size and both morphological and functional aspects of differentiation, the cells were classified as lower (a diameter <12.5 μM), middle (a diameter between 12.5 and 15 μM), and upper (a diameter >15 μM) epidermal cells. Fresh cells exposed to rhodaminated concanavalin A (Con A) were sedimented and reacted with fluoresceinated anti-Con A serum to distinguish cell surface Con A from intracellular lectin. Labeling at 4°C resulted in a uniform surface distribution of Con A irrespective of cell size. After a 1-hr incubation of Con A-labeled cells in lectin-free medium at 37°C, lower epidermal cells and approximately half of middle epidermal cells internalized Con A/receptor complexes by endocytosis while lectin remained diffusely on the remainder of middle epidermal cells and upper epidermal cells. By electron microscopy, ferritin-Con A was clustered on surface areas and invaginations of the plasma membrane before being endocytosed. We concluded that the differentiation of epidermal cells was accompanied by progressive decrease in endocytosis and, most probably, mobility of Con A receptors.     

Divergent actions of monomeric, dimeric, and tetrameric concanavalin A on lymphocyte traffic

The autoradiographic analysis of the localization of [³H]adenosine-labeled cells exposed to concanavalin A (Con A) in vitro has confirmed that the altered migration of Con A-treated lymphocytes is a consequence of their slower rate of migration and delay in normal areas of traffic (5, 6). The mechanisms through which Con A alters cell migration were further investigated by studying the effects of several derivatives of Con A on the distribution of ⁵¹Cr-labeled lymph node cells. The results obtained show that the monomeric and dimeric forms of Con A were unable to modify cell traffic, a condition that was partially reversed when succinyl Con A-treated cells were exposed to divalent antibodies to Con A. This suggests that Con A may alter lymphocyte recirculation by actively modifying the membrane fluidity or the surface lateral transport of the lymphocyte. Whatever the exact mechanisms responsible for the altered migration are, they probably involve complex active processes that can be related to the heterogeneity of Con A receptors, the existence of subsets of cells with different reactivities to the lectin, or simply the result of a passive phenomenon dependent on the presence of Con A on the cell surface.     

Radiation-induced alterations in binding of concanavalin A to cells and in their susceptibility to agglutination

Cell susceptibility to agglutination mediated by a plant lectin, concanavalin A (Con A), and the binding capacity of Con A to cells following gamma-irradiation have been examined in mouse myeloid leukaemia cells cultured in suspension. Irradiation caused an immediate decrease in the amount of Con A bound to the cell surface, whereas susceptibility of irradiated cells to agglutination by Con A was unchanged when compared to that of the unirradiated cells. Post-irradiation incubation of cells at 37 degrees C resulted in a temporary, more than 1.3-fold increase in cell susceptibility to agglutination 60 min after irradiation, whereas binding capacity of cells for Con A gradually recovered following irradiation, reaching a comparable level to that of unirradiated cells 3 h after irradiation. Cell susceptibility to agglutination by Con A does not depend strongly on its binding capacity.     

Glyceraldehyde-3-phosphate dehydrogenase is a major protein associated with the plasma membrane of retinal photoreceptor outer segments

A major 38-kDa protein associated with bovine rod outer segment plasma membranes, but not disk membranes, has been identified as glyceraldehyde-3-phosphate dehydrogenase on the basis of its N-terminal sequence and specific enzyme activity. This enzyme was extracted from lysed rod outer segments or isolated rod outer segment plasma membrane with 0.15 M NaCl and purified to homogeneity by affinity chromatography on a NAD(+)-agarose column. A specific activity of 90-100 units/mg of protein is within the range of activity obtained for glyceraldehyde-3-phosphate dehydrogenase isolated from other mammalian cells. Enzyme activity measurements indicate that this enzyme makes up approximately 2% of the total rod outer segment protein and over 11% of the plasma membrane protein. Protease digestion and binding studies on purified rod outer segment plasma and disk membranes suggest that glyceraldehyde-3-phosphate dehydrogenase reversibly interacts with a protease-sensitive plasma membrane-specific protein of rod outer segments. The finding that glyceraldehyde-3-phosphate dehydrogenase is present in large quantities in rod outer segments suggests that at least some of the energy required for the synthesis of ATP and GTP for phototransduction and other processes of the outer segment is derived from glycolysis which takes place within this organelle.     

Isolation of a concanavalin A receptor from mouse L cells.

A cell surface glycoprotein receptor for concanavalin A (Con A) has been isolated from mouse L cells. The isolation procedure involved dissolving whole L cells in 0.3 M lithium diiodosalicylate and extracting with aqueous phenol. The Con A receptor, which was found in the aqueous phase of this extract, was further purified by affinity chromatography on a column of Con A-Sepharose; the receptor was adsorbed to Con A-Sepharose and eluted with 0.1 M methyl alpha-D-glucopyranoside or with 0.1 M methyl alpha-D-mannopyranoside, but not with other monosaccharides. The cell surface location of the Con A receptor purified in this way was confirmed by showing that it can be isolated from purified L cell plasma membranes and by demonstrating that it can be labeled from the exterior surface of intact L cells by the nonpenetrating galactose oxidase-KB3H4 system. Biochemical studies of the Con A receptor have shown that it migrates on sodium dodecyl sulfate-polyacrylamide gels as a single component having an apparent molecular weight of approximately 100,000. Its N-terminal amino acid is valine and it has carbohydrate attached at several (at least five) different sites along the polypeptide chain.     

Phagocytosis of concanavalin A in normal and enucleated cultures of mammalian cells.

The fate of concanavalin A (Con A) bound to normal and enucleated L cells was followed at the ultrastructural level over a 20-h period. In both enucleates and normal cells the Con A is seen to be distributed in a uniform manner over the entire cell surface following a 30-min pulse with a low concentration of Con A. In the subsequent chase period the cells then aggregate the Con A and Con A sites into large clusters on the cell membrane. The cells then phagocytoze the Con A and large phagocytic vacuoles containing it are observed. Thus, enucleated cells are capable of phagocytozing Con A and its sites in the same manner as normal cells.     

Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label

We used fracture-label to establish ultrastructural localization of glycoproteins in cross-fractured nuclei of duodenal columnar and exocrine pancreatic cells. Mannose residues were detected in cell nuclei by labeling freeze-fractured tissues with concanavalin A-horseradish peroxidase X colloidal gold (Con A-HRP X CG) or direct concanavalin A X colloidal gold (Con A X CG); fucose residues were detected with Ulex Europaeus I X colloidal gold (UEA I X CG) markers. Areas of the three main intranuclear compartments (euchromatin, heterochromatin, and nucleolus) exposed by freeze-fracture were determined by automated image analysis. Colloidal gold particles bound to each nuclear subcompartment were counted and the results expressed in number of colloidal gold particles per square micrometer +/- SEM. Duodenal and pancreatic tissues fractured and labeled with Con A-HRP X CG complex or direct Con A X CG conjugates showed that the vast majority of Con A binding sites was confined to euchromatin regions with only sparse labeling of the heterochromatin and nucleolus. UEA I labeling of duodenal columnar cells showed that colloidal gold particles were almost exclusively confined to cross-fractured areas where euchromatin is exposed. Trypsinization of the fractured tissues before labeling with Con A and UEA I abolished 95-100% of the original label. Our results show that, within the nucleoplasm, mannose and fucose are residues of glycoproteins preferentially located within the regions of euchromatin.     

The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates

The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.     

Membrane differentiation of developing hemic cells of the bone marrow demonstrated by changes in concanavalin A surface labeling

The concanavalin A-gold labeled horseradish peroxidase (Con A-HRP-G) method has been employed in the ultrastructural localization of Con A surface receptor sites on glutaraldehyde-fixed normal human and guinea pig bone marrow cells. The number of gold particles per micron of cell surface was counted and data subjected to statistical analysis. All cells of the bone marrow exhibited Con A binding; however, the extent of surface labeling was dependent both on cell type and stage of differentiation. Distinctive modifications in mean surface density correlated with specific periods during the maturation of the erythrocytic, neutrophilic, eosinophilic and monocytic cell series. In several instances, the differentiative changes in surface Con A labeling proved to be species dependent. These observations are discussed in relationship to methodology and to potential changes in number and/or spatial arrangement of Con A receptor sites, primarily attributable to mannosyl and/or glucosyl residues associated with membrane glycoproteins and/or glycolipids of developing neutrophilic and erythrocytic cells.