NEBULIN AS AN ACTIN ZIPPER - A 2-MODULE NEBULIN FRAGMENT PROMOTES ACTIN NUCLEATION AND STABILIZES ACTIN-FILAMENTS

Nebulin is a family of giant muscle proteins (700-900 kDa) that intera ct with actin to form composite thin filaments in the skeletal muscle sarcomere. This modular protein is composed predominantly of repeating sequence modules of 31-38 residues. To understand the minimum size an d number of sequence modules that are required for actin interaction, we studied the behavior of a highly soluble two-module nebulin fragmen t ND8 that was expressed in Escherichia coli. By fluorescence spectros copy with pyrenyl-actin and co-sedimentation assays, we observed the f ollowing. 1) ND8 greatly accelerated actin nucleation, especially in a buffer that is suboptimal for actin nucleation. The presence of ND8 a bolished the lag phase of actin polymerization and increased the net e xtent of steady state polymerization, thereby reducing the critical co ncentration of actin polymerization. 2) ND8 reduced the rate of actin depolymerization and might increase the rate of elongation. 3) Cytocha lasin E, which caps both ends of actin filaments, inhibited the effect of ND8 on actin polymerization and caused the depolymerization of act in-ND8 complexes. These data suggest that ND8 interacts with actin in such a fashion that it stabilizes the actin nuclei and slows the depol ymerization from the ends of actin filaments. 4) The binding stoichiom etry of ND8 to F-actin, as estimated by co-sedimentation assays, is 1 to 2 mol of ND8 to 1 mol of actin with an apparent dissociation consta nt of 20 to 40 muM. Our data suggest that nebulin-actin interaction pr omotes actin nucleation and stabilizes preformed actin filaments, both of which are desirable attributes of a length-regulating template for actin filaments of the skeletal muscle. Each nebulin molecule may con tain as many as 100-200 actin binding domains to form a zipper-like ne bulin/actin composite filament.