Three cohesion molecules have been discovered in the slime mould Dictyostelium discoideum. Two of these molecules are involved in morphogenesis at the aggregation stage of the life cycle and thereafter, and may also provide an essential trigger for late gene expression. The third glycoprotein 126 (gp126) or contact site B, is present on axenically grown vegetative amoebae and persists to the aggregation stage where it is involved in the side-to-side cohesion of cells in aggregation streams. It is puzzling that vegetative amoebae should possess a cohesion molecule because their solitary habit does not necessitate cohesion. However, they do need to adhere to the substratum and adhesion of bacteria to the cells is a prerequisite for phagocytosis. Vogel et al. have proposed that the same receptor is involved in phagocytosis and cohesion. It has also been suggested that contact site B-mediated cohesion is a trigger for development. Using a specific antibody against gp126, we now show that contact site B is a phagocytosis receptor. Furthermore, contact site B is involved in regulating the size of aggregates formed during morphogenesis; it also seems to be involved in cell-substratum adhesion but is not a developmental trigger.
The developmental regulation of the vegetative cohesion molecule, gp126, has been monitored in the cellular slime mould Dictyostelium discoideum. As judged by immunoprecipitation using an anti-vegetative cell, cohesion blocking antibody, gp126 persisted until at least the grex stage of development although a decline in the level of the molecule was observed thereafter. Further, after the grex stage, cells showed an increasing loss of ability to absorb the cohesion-blocking effect of an anti-vegetative cell Fab. Therefore, the decline of gp126 could be ascribed to a loss from the cell surface. By radio-iodination of vegetative cells followed by liquid-scintillation counting of gp126, developmental-regulation could be determined quantitatively.At the grex stage of development, whole aggregates were embedded in wax. Longitudinal sections were then stained with a monospecific anti-gpl26 Fab, followed by a fluorescent sheep anti-rabbit IgG. Fluorescence was observed only at the tip (the prestalk) region, thereby showing that gp126 is a prestalk marker.To confirm the above result, grexes were dissociated and cells were separated into prespore and prestalk populations on a gradient of Percoll. Prestalk but not prespore cells were able to absorb the cohesion-blocking effect of an anti-vegetative cell Fab.To examine the biosynthesis of gpl26, cells were pulsed with radioactive glucosamine, mannose or acetate. The pattern of incorporation of radioactivity suggested that the de novo synthesis of gp126 ceases upon commencement of development.
Polyspecific antibodies were raised against vegetative cells of Dictyostelium discoideum, strain Ax2. Monovalent (Fab') fragments of antibodies CMC 1, 5, 7 and 12 blocked completely the cohesion of vegetative cells. Antibody CMC 1 was studied in detail. The Fab' of this blocked the cohesion of aggregation-competent cells by 40%. It also caused some loss of cell contact in aggregation streams. In so doing the contacts that remained were mostly at the ends of the cells. Immunofluorescence showed that CMC 1 Fab' bound to both the cytoplasm and the surface of fixed cells. It also bound to the surface of live cells. A control (N Fab') also bound to the cell surface but did not block vegetative cell cohesion. An extract of vegetative cells was obtained using the detergent Triton X-100. D. discoideum proteins were immunoprecipitated from this extract using protein A-Sepharose and CMC 1 immunoglobulin G (IgG). These immobilized proteins absorbed the cohesion-blocking activity of CMC 1 Fab'. About 30 proteins were obtained when the Triton-soluble fraction was immunoprecipitated with IgG of CMC 1, 5, 7 and 12. Five of these were found to be cell surface proteins by the technique of lactoperoxidase-catalysed radio-iodination. These proteins had molecular weights of 178 000, 166 000, 126 000 and 64 000. CMC 12 IgG immunoprecipitated an additional cell surface protein of 46 000 molecular weight. Slices of polyacrylamide gel containing each of the five proteins identified as possible contact sites were fixed, washed and incubated with CMC 1 Fab'. Gel that contained protein of 178 000, 166 000 and 64 000 molecular weight had no effect on the activity of CMC 1 Fab'. However, Fab' that had been incubated with gel containing protein of 126 000 molecular weight no longer blocked cell cohesion.
Dolichyl phosphate (C55) and dolichyl phosphate prepared from liver were incubated with an enzyme prepared from soya-bean protoplasts. They both stimulated the transfer of radioactivity from UDP-D-glucose to lipid, but the stimulation was greater with liver dolichyl phosphate. Liver dolichyl phosphate with the soya-bean enzyme stimulated the transfer of radioactivity from UDP-N-acetyl-D-[U-14C]glucosamine to acidic lipid. UDP-D-[U-14C]glucose and UDP-N-acetyl-D-[U-14C]glucosamine were used with the soya-bean enzyme to prepare the glycosylated-acid-lipid acceptor of each sugar. Mild acid hydrolysis revealed that the radioactivity in the lipid glucosylated from UDP-glucose was present exclusively as glucose. That in the lipid glycosylated from UDP-N-acetylglucosamine was present mostly as N-acetylglucosamine. The soya-bean acidic-lipid acceptors of glucose and N-acetylglucosamine were stable to both catalytic hydrogenation and treatment with hot aqueous phenol; they behaved in a similar way as authentic alpha-saturated glucosylated polyisoprenyl phosphates. The soya-bean acidic-lipid acceptors of glucose and N-acetylglucosamine were eluted as deoxycholate complexes from a Sephadex column. In comparison with glucosylated polyisoprenyl phosphates of known size, the patterns of elution indicated that both soya-bean lipids contained a polyisoprenoid chain of 18 isoprene units.
Protoplasts were prepared from cells of soya-bean (Glycine max) suspension cultures and the plasma membrane was labelled with diazotized [G-3H]sulphanilic acid. Homogenates were fractionated by differential and isopycnic centrifugation, and membrane fractions in a density gradient were characterized by enzymic markers and the radioactive label. When fractions containing a large amount of protein were incubated with UDP-[U-14C]glucose, radioactive material soluble in chloroform/methanol was formed and this separated into acidic and neutral fractions on ion-exchange chromatograms of DEAE-cellulose. The acidic fraction was shown to consist of dolichol phosphate glucose, and the neutral fraction sterol glucosides and acylsterol glucosides. Optimum conditions for glucosylation of dolichol phosphate were established as 5 mM-MgCl2, pH 6.0, and the enzyme had a Michaelis constant of 1.5 x 10(-5) m-UDP-glucose. Optimum conditions for glucosylation of sterol were 5 mM-MgCl2, pH 8.0 GDP-[U-14C]glucose was a poor substrate for the synthesis of both acidic and neutral lipids. Although the synthesis of dolichol phosphate glucose and sterol glucosides occurred throughout the sucrose gradient, the specific activities of both glucosyltransferases were greatest in a fraction coincident with the radioactively labelled plasma membrane. Results are discussed in relation to the likely role fo these transglucosylase activities.
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