DISRUPTION OF THE ACTIN CYTOSKELETON IN LIVING NONMUSCLE CELLS BY MICROINJECTION OF ANTIBODIES TO DOMAIN-3 OF CALDESMON

Two classes of affinity-purified polyclonal antibodies directed agains t the four different domains of gizzard caldesmon were prepared and th eir specificity was verified by immunochemical assays, One set of anti bodies recognized exclusively domain-3 spanning residues 483-578 and t he other one included IgG reactive toward the remaining domains-1, -2 and -4 corresponding to residues 1-482 and 581-756. Microinjection int o cultured fibroblasts of each antibody preparation was employed to pr obe the functional importance of the interaction between caldesmon and tropomyosin within living nonmuscle cells. Low concentrations of the antibody to domain-3 caused a rapid, severe and reversible disassembly of the microfilament network, In contrast, the antibodies to domains- 1, -2 and -4 were ineffective, The effects of the anti-domain-3 IgG we re observed not only with the purified antibody but also when present in the whole caldesmon antiserum serving for its isolation, The microf ilament disintegrating activity of this antibody was completely abolis hed upon preincubation with native caldesmon or a proteolytic caldesmo n fragment encompassing amino acids 483-578, In enzyme-linked immunoso rbent assay competition experiments, tropomyosin, but not F-actin, sig nificantly decreased the binding of the domain-3 antibody to caldesmon . Consistent with recent mutational studies pinpointing to the contrib ution of domain-3 in the in vitro binding of cellular caldesmon to tro pomyosin, the findings suggest that the microinjected domain-3 antibod y selectively disrupts the association of tropomyosin with caldesmon d omain-3, thereby destabilizing the protein complex and alleviating its known protective action against F-actin severing in the cell, Thus, o ur data further highlight the in vivo involvement of this particular d omain in the attachment of nonmuscle caldesmon to tropomyosin as well as the direct participation of the caldesmon-tropomyosin complex in th e cytoskeletal organization of the microfilaments.