ACTIN FILAMENT STRUCTURE PROBED WITH MONOCLONAL-ANTIBODIES

The interaction of two monoclonal antibodies (mAbs) with actin has bee n characterized to map die epitopes defined by these mAbs and to deter mine the accessibility of these sites in the actin filament (F-actin). Both mAbs react specifically with actin in radioimmunoassays and West ern blot assays, and by immunoprecipitation. The location of the epito pes within the primary structure of actin has been determined using li mited proteolysis of actin and Western blots, or using immunoprecipita tion of truncated actin fragments synthesized in a cell free translati on assay. Both mAbs bind to the C-terminal fragment of actin (residues 68375) produced by chymotrypsin cleavage. One epitope is further loca lized to a 9.9 kD peptide corresponding to residues 5-93. Therefore, t he epitope defined by this mAb (2G11.4) lies between residues LyS6g an d Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal o f residues 356-365 from the C-terminus of actin completely abolished t he binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of the se epitopes in native F-actin was determined with solution binding ass ays and characterized by immunoelectron microscopy. Monoclonal antibod y 4E3.adl binds strongly to filaments, resulting in bundling or decora tion of F-actin depending on the valency of the mAb, and indicating th at the epitope is readily accessible in F-actin. In contrast, mAb 2G11 .4 disrupts F-actin structure, resulting in the formation of an amorph ous immunoprecipitate. These results place constraints on models of ac tin filament structure.