Identification of the cohesion molecule, contact sites B, of Dictyostelium discoideum

Abstract

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.