Method for macromolecular colocalization using atomic recombination in dynamic SIMS.

Localizing two or more components of assemblies in biological systems requires both continued development of fluorescence techniques and invention of entirely new techniques. Candidates for the latter include dynamic secondary ion mass spectrometry (D-SIMS). The latest generation of D-SIMS, the Cameca NanoSIMS 50, permits the localization of specific, isotopically labeled molecules and macromolecules in sections of biological material with a resolution in the tens of nanometers and with a sensitivity approaching in principle that of a single protein. Here we use two different systems, crystals of glycine and mixtures of proteins, to show that the formation of recombinant CN secondary ions under Cs bombardment can be exploited to create a new colocalization technique. We show experimentally that the formation of the recombinant (13)C(15)N secondary ion between (13)C- and (15)N-labeled macromolecules is indeed an indicator of the distance between the interacting macromolecules and on their shape. We build up a convolution model of the mixing-recombination process in D-SIMS that allows quantitative interpretations of the distance-dependent formation of the recombinant CN. Our results show that macromolecules can be colocalized if they are within 2 nm of one another. We discuss the potential advantages of this new technique for biological applications.

Quantitative and qualitative approach of glycan-glycan interactions in marine sponges.

Cell recognition and adhesion involving many kinds of cell surface molecules operate via homotypic and/or heterotypic protein-protein and protein-carbohydrate binding. Our investigations in marine sponges have provided direct evidence for a novel molecular mechanism of multivalent glycan-glycan binding related to cellular interactions. Biochemical characterization of purified proteoglycans revealed the presence of specific acidic glycans, different from classical glycosaminoglycans. Such acidic glycans of high molecular weight, containing fucose, glucuronic or galacturonic acids, and pyruvate and sulfate groups may represent a new class of primordial proteoglycans, named by us glyconectins. The thermodynamic and kinetic approaches of biological macromolecule interactions do not provide a direct measurement of the intermolecular binding forces that are fundamental for the function of the ligand-receptor association. Using the atomic force microscopy (AFM), we provided the first quantitative evaluation of the binding strength between cell adhesion proteoglycans. Measurement of binding forces intrinsic to cell adhesion glyconectin proteoglycans (AGPs) is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. (i) As a model, we selected the cell AGP isolated from the marine sponge Microciona prolifera; it mediates in vivo cell recognition and aggregation via homotypic, species-specific, multivalent, and calcium ion-dependent glycan-glycan interactions. (ii) Under physiological conditions, a large cohesive force theoretically able to hold the weight of approximately 1600 cells was measured. (iii) The C-2 autocomplementarity model for AGP-AGP interactions; and (iv) the requirement of the calcium ionic bridges suggest also that the self-recognition and multivalency of glycan-glycan interactions are essential for cell adhesion. (v) The evolution of glyconectin-like proteoglycan molecules may have been a fundamental prerequisite for the emergence of the first multicellular organisms. Glycan-glycan interactions may thus provide a new paradigm for molecular self-recognition.

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions.

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteoglycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today's simplest living Metazoa), are proposed to be the primary cell adhesive molecules essential for the evolution of the multicellularity.

Molecular self-recognition and adhesion via proteoglycan to proteoglycan interactions as a pathway to multicellularity: atomic force microscopy and color coded bead measurements in sponges.

During the emergence of multicellular organisms, molecular mechanisms evolved to allow maintenance of anatomical integrity and self-recognition. We propose that carbohydrates from proteoglycans, as the most peripheral cell surface, and matrix molecules might have provided these key adhesion and recognition functions. If so, the Porifera as the simplest metazoans alive today should retain, at least in part, proteoglycan adhesion and recognition mechanisms. Early work on cell adhesion of dissociated marine sponge cells provided important phenomenological evidence for cell sorting. Here is reviewed recent work on molecular mechanisms of cell recognition and adhesion mediated by cell surface proteoglycans purified from three marine sponge species, Microciona prolifera, Halichondria panicea, and Cliona celata. Biochemical characterization of isolated proteoglycans showed that each species expressed a unique type of primordial molecule named glyconectins. These proteoglycans displayed species-specific self-recognition and adhesion in color-coded bead, cell, and blotting assays. The specificity of homophilic proteoglycan to proteoglycan interactions in the Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily system. Such xeno-selectivity may be a new paradigm for the molecular self-recognition, which was a fundamental requirement in the self/non-self discrimination during the emergence of multicellularity and further divergence of species. We have used atomic force microscopy (AFM) technology to directly measure intermolecular binding strength between individual pairs of ligand and receptor molecules in physiological solution. Homophilic glyconectin interactions were investigated by AFM after covalent attachment of the protein core to the sensor tip and to a flat surface, leaving the carbohydrates unmodified. AFM measurements of the binding strength between glyconectins indicated that one pair of molecules could theoretically hold the weight of 1,600 cells in physiological solution. These results provided the first essential and quantitative evidence that proteoglycan-proteoglycan binding can perform the adhesion function that we have assigned to it. Our investigations with purified proteoglycans from the marine sponge M. prolifera (glyconectin 1) using bead and cell adhesion assays have provided evidence that a new molecular mechanism of polyvalent and specific glycan-glycan binding between proteoglycans can mediate cell recognition and adhesion. Partial sequencing of the glycans has revealed two new cell adhesion carbohydrate structures: (3)GlcNAc(3OSO3)beta1-3Fuc and Pyr4,6Galbeta1-4GlcNAcbeta1-3Fuc.

PDGF-BB increases endothelial migration on cord movements during angiogenesis in vitro.

To explore direct effects of platelet-derived growth factor (PDGF) on endothelial cells during angiogenesis in vitro, we have used cloned bovine aortic endothelial cells that spontaneously form cord structures. Recently we have shown that cells forming these endothelial cords express PDGF beta-receptors and that PDGF-BB can contribute to cellular proliferation and cord formation. In this study we investigated whether PDGF-induced cellular migration might also contribute to endothelial repair and angiogenesis in vitro. Ten individual endothelial cells in cords were tracked at an early stage of cord formation by video-timelapse microscopy. PDGF-BB (100 ng/ml) induced an increase in endothelial cell movement of 67 +/- 15% as compared with diluent control. Interestingly, PDGF-BB also increased movements of entire cord structures, followed at branching points, by 53 +/- 12% over diluent control. Taken together, these video-timelapse experiments suggested that the apparent movements of single endothelial cord cells might also be due to the motion of entire underlying cord structures in response to PDGF. To analyze the response of single endothelial cord cells we therefore examined whether PDGF-induced migration contributes to endothelial repair. Abrasions were applied with a razor blade to confluent monolayers of endothelial cells at an intermediate stage of cord formation. PDGF-BB concentration-dependently increased the distance to which cord-forming endothelial cells migrated into the abrasion. An increased number of elongated, i.e., probably migrating, endothelial cells was found in the abrasion in response to PDGF-BB. However, there was no effect of PDGF-BB on the total number of endothelial cells found in the abrasion. PDGF-AA affected neither the distance to which the cells migrated nor the number of elongated cells. Actin and tubulin stainings revealed that these cytoskeletal structures were not appreciably altered by PDGF-BB. Furthermore, urokinase-type plasminogen activator transcripts were not modulated in response to PDGF-BB. We conclude that in this model of angiogenesis in vitro PDGF-BB can elicit the movement of entire cord structures, possibly via u-PA-independent mechanisms. PDGF-BB also controls the migration of single cord-forming endothelial cells. Thus, PDGF-BB possibly contributes to endothelial repair and angiogenesis by direct effects on proliferation and composite movements of PDGF beta-receptor-expressing endothelial cells and cords.

Characterization of a novel sulfated carbohydrate unit implicated in the carbohydrate-carbohydrate-mediated cell aggregation of the marine sponge Microciona prolifera.

Species-specific cell reaggregation in the marine sponge Microciona prolifera is mediated by an adhesion proteoglycan. Two interactions are involved in the process: a Ca(2+)-dependent homophilic binding between proteoglycan molecules and a Ca(2+)-independent binding between the molecule and cells. Both interactions are mediated by the glycan moieties of the proteoglycan. The interaction of the proteoglycan with itself has been characterized as a carbohydrate-carbohydrate interaction of multiple low affinity sites. The monoclonal antibodies Block 1 and Block 2 raised against the purified aggregation proteoglycan and selected for inhibition of aggregation bind to these glycans. In a previous report the structure, [formula: see text] was assigned to the oligosaccharide reacting with Block 1 antibody (Spillmann, D., Hård, K., Thomas-Oates, J., Vliegenthart, J. F. G., Misevic, G., Burger, M. M., and Finne, J. (1993) J. Biol. Chem. 268, 13378-13387). By the technique of attaching the water-soluble acid-degraded fragments to a lipid carrier for immunochemical detection and by chemical, enzymatic and spectroscopic methods the structure, [formula: see text] was assigned to the oligosaccharide reacting with the aggregation-blocking monoclonal antibody Block 2. The structure, [formula: see text] was assigned to a major nonreactive oligosaccharide, which outlined the molecular requirements of antibody binding of the two aggregation-associated epitopes. These data demonstrate that two different functional sites with distinct structural characteristics and antibody reactivities are involved in the reaggregation of sponge cells, a model of carbohydrate-carbohydrate-mediated cell interactions.

Binding strength between cell adhesion proteoglycans measured by atomic force microscopy.

Measurement of binding forces intrinsic to adhesion molecules is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. Atomic force microscopy was used to measure the binding strength between cell adhesion proteoglycans from a marine sponge. Under physiological conditions, the adhesive force between two cell adhesion molecules was found to be up to 400 piconewtons. Thus a single pair of molecules could hold the weight of 1600 cells. High intermolecular binding forces are likely to form the basis for the integrity of the multicellular sponge organism.

A novel class of embryonic cell adhesion glycan epitopes is expressed in human colon carcinomas.

A new class of acidic glycans isolated from marine sponges and sea urchin embryos was shown to mediate cell adhesion via carbohydrate-carbohydrate interactions. Cell aggregation blocking monoclonal antibodies (Block 1 and Block 2 mAbs) directed against these polysaccharides localized functional epitopes in embryonic sea urchin gut. Immunofluorescence light microscopy of human colon carcinomas and healthy colon samples with Block 1 and Block 2 mAbs established that the carbohydrate structures similar to the invertebrate acidic glycan adhesion molecules are also expressed in humans. The Block 1 mAb labeled basal and apical lamina of tumor cells, whereas the Block 2 bound exclusively to the apical part of the epithelium. In normal tissue whole goblet cell membrane was stained with both antibodies indicating that transformation leads to spatial rearrangement of glycan antigens. Acidic glycans from human colon carcinomas and normal colon were isolated after delipidation, and complete pronase and DNase digestion, by gel filtration and adsorption to anion exchange membranes. Immunodot assay with Block 1 and Block 2 mAbs revealed that tumor cells have elevated expression of both carbohydrate structures. These results suggest that the acidic glycan adhesion molecules, originally found in sponges and sea urchin embryos, may represent a new class of carbohydrate carcino-embryonal antigens involved in cellular interactions associated with morphogenesis, metastasis and renewal of adult tissue.

Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO4(2-) uptake and expression of a band 3 like protein in the marine sponge, Microciona prolifera.

Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: 1) impairment of aggregation, 2) loss of cell movements, 3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of 35SO4(2-) uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in 35SO4(2-) uptake and incorporation which could be greatly augmented in the presence of Ca2+/Mg2+. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa 35SO4(2-) sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.

A novel class of adhesion acidic glycans in sea urchin embryos. Isolation, characterization and immunological studies during early embryonal development.

Total glycans were isolated and purified from Lytechinus pictus embryos at early developmental stages by gel-filtration chromatography after pronase and DNase digestion, and alkali-borohydride treatment. Fractionation by Superose 6 and HPLC gel-filtration chromatography revealed three major glycan fractions of 580, 150 and 2 kDa consistently throughout development up to the stage of end gastrula. The 580-kDa and the 150-kDa glycan fractions isolated from fertilized eggs up to the stage of end gastrula are highly acidic, whereas the 2-kDa glycan fractions have no detectable uronic acid residues and charged groups. Chemical analysis of the glycan fractions showed that their content of neutral hexoses, uronic acid, GlcNAc, GalNAc and sulphate changes during development. The resistance of the 580-kDa and the 150-kDa glycan fractions to glycosaminoglycan-degrading enzymes indicates a structure which is different from the glycosaminoglycans. The incorporation of [3H]glucosamine into the 580-kDa, the 150-kDa and the 2-kDa glycan fractions showed that glycan synthesis increases in a linear fashion from the stage of early blastulation to end of gastrulation. Maximal incorporation of the radioligand occurs in the 2-kDa glycan fractions, with the highest rate observed between the stages of mesenchyme blastula and early gastrulation. Immunological studies, using a monoclonal antibody which inhibits cell aggregation, showed that the total glycans isolated from morula, early blastulation, early gastrulation and the end of gastrulation carry cell-adhesion epitopes. The number of these epitopes, as indicated by the intensity of the immunostaining, increases from morula formation to end-gastrulation stages and correlates with the increased rate of morphogenetic movements. These results suggest that controlled expression of the cell-adhesion glycan epitopes play an important role in sea urchin gastrulation.

Isolation and characterization of a new class of acidic glycans implicated in sea urchin embryonal cell adhesion.

Three major glycan fractions of 580 kDa (g580), 150 kDa (g150), and 2 kDa (g2) were isolated and purified from Lytechinus pictus sea urchin embryos at the mesenchyme blastula stage by gel filtration and high pressure liquid chromatography. Chemical analysis, by gas chromatography, revealed that g580 is highly sulfated and rich in N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, and fucose. The g150 fraction is less acidic than g580 and contains high amounts of amino sugars, xylose, and mannose. The g2 fraction is neutral, rich in N-acetylglucosamine, mannose, and galactose. The g580 and g150 fractions are resistant to glycosaminoglycan-degrading enzymes, indicating that they are distinct from the glycosaminoglycans. The g580 fraction resembles, with respect to chemical composition, a previously characterized 200 kDa sponge adhesion glycan (g200). The binding of the monoclonal antibody Block 2, which recognizes a repetitive epitope on g200, as well as of the anti-g580 polyclonal antibodies to both g580 and g200 indicated that these two glycans share similar antigenic determinants. The Fab fragments of the Block 2 antibody, which previously have been shown to inhibit cell adhesion in sponges, also blocked the reaggregation of dissociated sea urchin mesenchyme blastula cells. These results indicate that g580 carries a carbohydrate epitope, similar to the sponge adhesion epitope of g200, which is involved in sea urchin embryonal cell adhesion.

Characterization of a novel pyruvylated carbohydrate unit implicated in the cell aggregation of the marine sponge Microciona prolifera.

The species-specific Ca(2+)-dependent reaggregation of dissociated cells of the marine sponge Microciona prolifera is mediated by a large extracellular adhesion proteoglycan. The glycans of this molecule are involved in the interactions of the proteoglycan with itself and with the sponge cells. Monoclonal antibodies against the glycans block the aggregation of sponge cells (Misevic, G. N., Finne, J., and Burger, M. M. (1987) J. Biol. Chem. 262, 5870-5877). Proteoglycan oligosaccharides were prepared by partial acid hydrolysis of the isolated glycans, and their reactivity with the monoclonal antibodies was monitored after linkage to phospholipid and immunostaining of thin layer chromatograms. One major antibody-reactive oligosaccharide was detected and purified by ion-exchange chromatography and high performance liquid chromatography. 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, methylation analysis, and sequential chemical and enzymatic degradation studies indicated the structure [formula: see text] for the oligosaccharide. The depyruvylated derivative of the oligosaccharide did not react with the aggregation-blocking antibody, which indicates that the pyruvate acetal is an essential part of the epitope.

Expression of type IV collagen-degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis.

Type IV collagen-degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600-fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non-collagenous proteins of the extracellular matrix. Chelators such as 1,10-phenanthroline or Na2EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen-degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus-larva stage. The specific inhibitors of collagen biosynthesis 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide and tricyclodecane-9-yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen-degrading activity to non-detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen-degrading activity.

Carbohydrate-carbohydrate interactions of a novel acidic glycan can mediate sponge cell adhesion.

Cell recognition and adhesion were first demonstrated in marine sponges. These phenomena were later shown in Microciona prolifera sponge to be mediated by a Ca(2+)-dependent self-association of adhesion proteoglycans (APs) attached in a species-specific manner to cell-surface receptors. Using the same experimental system we now provide three lines of evidence that highly polyvalent Ca(2+)-dependent carbohydrate-carbohydrate interactions of a novel AP glycan represent the basis of AP-AP self-binding and thus of cell adhesion. 1) A specific monoclonal antibody which blocks cell aggregation and AP bead adhesion identified a highly repetitive novel carbohydrate epitope (2500 sites) in an acidic glycan of M(r) = 200 x 10(3) (g200) from AP. 2) Reconstitution of the Ca(2+)-dependent self-interaction activity of AP was achieved by cross-linking the purified protein-free g200 glycan into polymers of similar valency as the native AP. 3) Beads coated with the protein-free g200 glycan showed a Ca(2+)-dependent aggregation equivalent to that of AP beads. Carbohydrate and amino acid analyses of the g200 glycan purified by gel electrophoresis, high performance liquid chromatography gel filtration, and ion exchange chromatography yielded six components in the following proportions; 68 fucose, 32 glucuronic acid, 2 mannose, 18 galactose, 19 N-acetylglucosamine, and 1 asparagine residue. These unique chemical features together with immunological and enzymological analyses suggest that the g200 glycan is a large highly fucosylated, acidic, N-linked polysaccharide with a novel structure distinct from that of other known glycosaminoglycans.