Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation-Defective Mutants of Dictyostelium discoideum

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Abstract
The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two‐stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with 125I‐Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread‐sorption of the antiserum with discoidin‐Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified 125I‐discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells. These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation‐defective mutants. Strain EB‐32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB‐18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.