The actin cytoskeleton is regulated by a variety of actin-binding proteins including those constituting the tropomyosin family. Tropomyosins are coiled-coil dimers that bind along the length of actin filaments. In muscles, tropomyosin regulates the interaction of actin-containing thin filaments with myosin-containing thick filaments to allow contraction. In nonmuscle cells where multiple tropomyosin isoforms are expressed, tropomyosins participate in a number of cellular events involving the cytoskeleton. This chapter reviews the current state of the literature regarding tropomyosin structure and function and discusses the evidence that tropomyosins play a role in regulating actin assembly.

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of crosslinking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERKphosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys 595 , away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.