Measuring Mechanodynamics using an Unsupported Epithelial Monolayer Grown at an Air-Water Interface

Actomyosin contraction and relaxation in a monolayer is a fundamental biophysical process in development and homeostasis. Current methods used to characterize the mechanodynamics of monolayers often involve cells grown on solid supports such as glass or gels. The results of these studies are fundamentally influenced by these supporting structures. Here, we describe a new methodology for measuring the mechanodynamics of epithelial monolayers by culturing cells at an air-liquid interface. These model monolayers are grown in the absence of any supporting structures allowing us to remove cell-substrate effects. This methods potential was evaluated by observing and quantifying the generation and release of internal stresses upon actomyosin contraction (320{+/-}50Pa) and relaxation (190{+/-}40Pa) in response to chemical treatments. This is in contrast to the results observed in monolayers grown on solid substrates (glass and gels) where movement was drastically muted. Tracking the displacement of cell nuclei, cell edges and cluster perimeter allowed us to quantify the strain dynamics in the monolayer indicating the reliability of this method. New insights were also revealed with this approach. Although unsupported monolayers exhibited clear major and minor strain axes, they were not correlated to the general alignment of cell nuclei. The situation was dramatically different when the monolayers were grown on soft gels and hard glass substrates. It was observed that both gels and glass substrates led to the promotion of long-range alignment of cell nuclei. In addition, the strain orientation was correlated to nuclear alignment on the soft deformable gels. This new approach provides us with a picture of basal actomyosin mechanodynamics in a simplified system allowing us to infer how the presence of a substrate impacts actomyosin contractility and long-range multi-cellular organization and dynamics. This new methodology will also enable many new questions to be asked about the molecular regulation of the mechanodynamics of unsupported monolayers.