Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin

Abstract

In striated muscle the extent of the overlap between actin and myosin filaments contributes to the development of force. In slow twitch muscle fibers actin filaments are longer than in fast twitch fibers, but the mechanism which determines this difference is not well understood. We hypothesized that tropomyosin isoforms Tpm1.1 and Tpm3.12, the actin regulatory proteins, which are specific respectively for fast and slow muscle fibers, differently stabilize actin filaments and regulate severing of the filaments by cofilin-2. Using in vitro assays, we showed that Tpm3.12 bound to F-actin with almost 2-fold higher apparent binding constant (Kapp) than Tpm1.1. Cofilin2 reduced Kapp of both tropomyosin isoforms. In the presence of Tpm1.1 and Tpm3.12 the filaments were longer than unregulated F-actin by 25% and 40%, respectively. None of the tropomyosins affected the affinity of cofilin-2 for F-actin, but according to the linear lattice model both isoforms increased cofilin-2 binding to an isolated site and reduced binding cooperativity. The filaments decorated with Tpm1.1 and Tpm3.12 were severed by cofilin-2 more often than unregulated filaments, but depolymerization of the severed filaments was inhibited. The stabilization of the filaments by Tpm3.12 was more efficient, which can be attributed to lower dynamics of Tpm3.12 binding to actin.

Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments

“…First, we checked if the digestion rate of wild type homodimer Tpm2.2 differed from the digestion rate of the crosslinked wild type homo- and heterodimers. As observed previously [ 34 ], free tropomyosin was completely digested within 3 min, but dimers were partially protected by F-actin ( Figure 4 A). Crosslinking the chains of Tpm2.2 slowed down the initial rate of proteolysis, but crosslinked Tpm1.1/Tpm2.2 heterodimer was digested at a rate indistinguishable from the noncrosslinked homodimer ( Figure 4 B).…”

“…Free tropomyosin is easily digested by trypsin, but this process is partially inhibited by F-actin [ 33 ]. Because the time course of digestion depends on the tropomyosin sequence [ 34 ], the rate of trypsin digestion may be indicative of changes to tropomyosin dynamics due to heterodimer formation or amino acid substitutions.…”

“…How susceptible tropomyosin is to trypsinolysis provides insight into any structural heterogeneity of homo- and heterodimers of wild type and mutant Tpm2.2. Free tropomyosin is cleaved very quickly, but is partially protected by F-actin [ 33 , 34 ]. The differences in the proteolysis rate between tropomyosin variants can be explained by their specific dynamics or orientation, which may either expose or hide the peptide bonds recognized by trypsin.…”

CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

“…In striated muscle, actin filaments are strongly protected against disassembly by regulatory proteins, including Tpm. Tpm1.1 inhibits the depolymerization activity of CFL2 63 and also CAP2 (shown above). Therefore, we hypothesize that CAP2 is responsible for accelerating cofilin-mediated depolymerization of a dynamic population of non-regulated actin filaments, potentially comprised of α-SMA and α-SKA, that are not stabilized by Tpm at their pointed ends.…”

Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin

“…The isoforms are products of TPM1, TPM2, and TPM3 genes, respectively [3]. Although Tpm1.1 and Tpm3.12 are highly homologous proteins showing 91% identity and ~96% sequence similarity [4], their regulatory functions are quantitatively differentiated [5,6]. However, it is not known, which sequence differences are responsible for the regulatory performance of the isoforms.…”