Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin

Abstract

The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for α-actin (αA), α-actin-tropomyosin-αβ (αA-Tmαβ), α-actin-tropomyosin-β (αA-Tmβ), γ-actin (γA), γ-actin-tropomyosin-αβ (γA-Tmαβ), and γ-actin-tropomoysin-β (γA-Tmβ). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of αA, γA, and γA-Tmαβ is not distinguishable. Compared to these three 'baseline conditions', statistically significant differences (p<0.05) were: αA-Tmαβ - actin sliding velocity increased 1.12-fold, γA-Tmβ - motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, αA-Tmβ - run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: αA-Tmαβ - myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, γA-Tmβ - mechanical coupling between myosins is stronger, αA-Tmβ - the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics. © 2013 Hilbert et al.