Selection of Dictyostelium mutants defective in cytoskeletal proteins: use of an antibody that binds to the ends of alpha-actinin rods.
A monoclonal antibody, mAb 47-19-2, was used to study the subunit topology of the rod-shaped alpha-actinin molecules of Dictyostelium discoideum and to screen for mutants defective in the production of alpha-actinin. Electron microscopy of rotary-shadowed alpha-actinin-antibody complexes showed binding of mAb 47-19-2 to both ends of the alpha-actinin rods and cleavage of the rods into its subunits, indicating that the two subunits of alpha-actinin extend in an anti-parallel mode through the whole length of the rod. The antibody binding sites were located in close proximity to the sites responsible for actin cross-linking, which is consistent with the blocking activity of the antibody. In a mutant, HG1130, no antibody label was detected in colony blots, and by immunoblotting of mutant proteins separated by SDS-PAGE, only trace amounts of alpha-actinin were found. The mutant showed normal binding of antibodies directed against the actin-binding proteins severin and capping protein. The mutation responsible for the alpha-actinin defect was recessive and located on linkage group I of the genetic map of D. discoideum. HG1130 cells grew on bacteria at a normal rate and also axenically like cells of the parent strain AX2. After starvation the mutant cells expressed the contact site A glycoprotein, a marker of the aggregation-competent stage, and reacted chemotactically to cyclic AMP. The aggregation patterns and fruiting bodies of the mutant appeared to be normal. Patching and capping on the surface of HG1130 cells was induced by antibodies against the contact site A glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)
D000185:Actinin / Q000235:genetics* / Q000276:immunology; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002465:Cell Movement; D002633:Chemotaxis; D003598:Cytoskeletal Proteins / Q000235:genetics*; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development; D009154:Mutation; D011487:Protein Conformation; D013045:Species Specificity
D000906:Antibodies; D000936:Antigen-Antibody Complex; D003598:Cytoskeletal Proteins; D000185:Actinin