Methods Cell Biol 2015 Jan 01;128:83-103. doi: 10.1016/bs.mcb.2015.02.001.

Reconstituting cytoskeletal contraction events with biomimetic actin-myosin active gels.

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The actin-myosin cytoskeleton allows cells to move, change shape, and exert forces. These fascinating functions involve active contraction of cross-linked networks of actin filaments by myosin II motor proteins. Unlike muscle cells, where actin and myosin form ordered bundles that contract homogeneously, nonmuscle cells have a variety of more disordered types of actin-myosin meshworks. Active gels reconstituted from purified actin and myosin proteins offer a useful in vitro model system to systematically and quantitatively investigate the mechanisms of contraction and the role of physical parameters like motor activity and network connectivity. In order to quantify the effect of these physical parameters on contraction, time-lapse microscopy combined with quantitative image analysis is required. Here we describe an assay that we developed specifically to record contraction events of entire biomimetic active gels in contraction chambers, which enables one to systematically quantify the dependence of contraction time and length scales on experimental parameters such as protein concentrations, adenosine triphosphate concentration, ionic strength, and surface adhesion.

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Genes referenced: cep152 LOC590297