Abstract
Much is known regarding the structure and function of contractile actomyosin networks in cellular physiology, however, details of their biophysical properties remain far from clear. For example, we lack a clear understanding of how the transmission of forces from myosin motor filaments influences structural changes in dynamic actin networks. We attempt to address these unknowns by measuring the dynamic structure and biophysical properties of in vitro 2-D actomyosin bundles. By working with a small number of purified components, this enables us to create a simple assay to study the effects of small changes in concentrations of one component has on the emergent biophysical properties of the resultant contractile bundle. Here we have created a reconstituted 2-D network of actin that is suspended from, and anchored to, a surface using polystyrene beads. Smooth muscle myosin (ADP) is added, resulting in bundling of actin within the network. Interestingly, the resultant structure after myosin addition allows inter-actin bundling to occur, creating a web-like structure. The addition of ATP initiates contraction and results in large scale restructuring of the actin bundles. During network contraction, the intensity of individual actin bundles increases as the individual filament arc length decreases. In addition, the web-like structure of the network diminishes during contraction. Under some conditions, presumably where the myosin/actin ratio is above a critical threshold, the filaments break due to excessive contraction. These tethered filaments, no longer under tension, contract at a greater rate than those still anchored by both ends. In conclusion, actomyosin filaments can be assembled in vitro without a passive crosslinker; ADP/ATP myosin can bundle actin, even during contraction. Large-scale restructuring of 2-D actomyosin networks occur when tension is applied through myosin motor activity. These observations are consistent with sliding filament theory of actomyosin contraction.
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