Abstract
Tropomyosins are a family of ubiquitously expressed actin binding proteins that bind along the length of actin filaments, and have established roles in stabilizing their dynamics and governing myosin motor activity. Although Tropomyosin was one of the first actin associated proteins discovered, its role in regulating actin filament dynamics is still poorly understood. Saccharomyces cerevisiae expresses two Tropomyosin isoforms, Tpm1 and Tpm2, which have traditionally been thought to play redundant roles in stabilizing actin cables. However, strong genetic evidence collected by our lab shows that TPM2, but not TPM1, may destabilize rather than stabilize actin networks in vivo. In light of this evidence, I performed complementary in vivo and in vitro tests of Tpm2 function designed to elucidate its specific roles in regulating actin filament dynamics. My in vivo analysis confirmed that TPM2 helps prevent overgrowth of actin cables, consistent with a role in either destabilizing actin filaments or suppressing actin filament growth. However, in vitro analysis showed that purified Tpm2 has no effect on actin filament stability, and in fact appears to weakly protect against cofilin-mediated severing. Instead, Tpm2 showed potent, formin-specific effects on actin filament nucleation that may explain its in vivo roles. Specifically, Tpm2 strongly inhibited nucleation by the formin Bnr1, yet enhanced nucleation by the formin Bni1. These results suggest a novel isoform-specific activity of Tpm2 in regulating actin cable formation in vivo, and provide the first evidence of a Tropomyosin controlling actin nucleation, and the first evidence for Tropomyosins differentially regulating the functions of formins.