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

Lennart Hilbert,Anne-Marie Lauzon,Horia N Roman,Jenna L Blumenthal,Michael C Mackey,Nedjma B Zitouni,Apolinary Sobieszek,Genevieve Bates,Andrew D Mcculloch

doi:10.1371/journal.pcbi.1003273

Lennart Hilbert, Anne-Marie Lauzon + Show 7 more

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https://doi.org/10.1371/journal.pcbi.1003273

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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 -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). 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 A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – 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: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – 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.

Highlights

Smooth muscle contractile protein expression Differential expression of smooth muscle contractile proteins has been associated with organismal development [1], contractile phenotypes [2,3,4], and pathologies, e.g. preterm labour, hypertrophic bladder, or airway hyper-responsiveness [5,6,7]
We found that the actin isoforms cause differences in the mechanical interaction only when tropomyosin is present, not without it
All different actin-tropomyosin combinations affected the mechanical interactions in a different way

Summary

Introduction

Smooth muscle contractile protein expression Differential expression of smooth muscle contractile proteins has been associated with organismal development [1], contractile phenotypes [2,3,4], and pathologies, e.g. preterm labour, hypertrophic bladder, or airway hyper-responsiveness [5,6,7]. While the role of the smooth muscle myosin isoforms has been extensively investigated [7,8,9], the functional implications of the differential expression of specific actin and actin-regulatory protein isoforms remain elusive [4]. Smooth muscle actin In smooth muscle, actin isoforms are expressed from four different genes, yielding ‘‘vascular muscle’’ a- and ‘‘enteric muscle’’ c-actin, as well as non-muscle (cytoplasmic) a- and c-actin. The muscle isoforms are associated with the contractile apparatus, the non-muscle isoforms with cytoskeletal structures [5]. Muscle a-actin is generally associated with tonic, c-actin with phasic smooth muscles [5,10,11]. Diseaserelated expression differences in a- vs c-actin have been found [6]. Insight from tissue level mechanics seems lacking, too [4]

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