Tropomyosin Phosphorylation Has Filament-Level And Crossbridge-Level Effects On Actin-Myosin Interactions

Abstract

The α-isoform of tropomyosin (Tm) can be phosphorylated near the C-terminus, and dephosphorylation of Tm has been shown to decrease actin-myosin ATPase rates (Heeley et al., 1989). Additionally, Tm polymerizes with adjacent Tm molecules in a head-to-tail manner. Phosphorylation of Tm might therefore influence the strength of these interactions, and thus regulate the degree of cooperative activation by myosin binding. We tested Tm phosphorylation effects by measuring the force and velocity of actin-myosin interactions at the level of single actin filaments using a combination of actin-Tm binding assays, in vitro motility assays, and a novel, high throughput laser trap assay to measure isometric force. In these assays we used purified heavy meromyosin (HMM) and actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. Dephosphorylation of Tm did not result in significant changes in actin-Tm binding or unloaded sliding velocities. Isometric force measurements showed that thin filaments were cooperatively activated by myosin, but only when Tm was phosphorylated. When Tm was dephosphorylated, the reconstituted filaments behaved like bare actin filaments. These results suggest transmission of cooperative activation beyond one thin filament regulatory unit when Tm is phosphorylated. Moreover, Tm phosphorylation increased isometric force production ∼50% compared to bare actin filaments at intermediate HMM surface densities. In combination with sliding velocity data, this result suggests that Tm phosphorylation may have effects at the level of a single crossbridge that cannot be explained by steric hindrance. We hypothesize that Tm phosphorylation accelerates the rate of crossbridge attachment. Single molecule kinetic and force spectroscopy experiments are underway to gain insight on this possibility.