Boundary-maintenance mechanism in cell segregation in vitro

Abstract

Embryogenesis has long been shown to involve an active rearrangement of cells by migration and segregation. Once the segregation of different cell types is established, cells continue to proliferate, exerting compressive stresses to the neighboring cells. Meanwhile, the cells are constantly challenged to maintain their segregated layers. Several studies have shown the formation of an actomyosin-driven boundary-like structure that prevents the intermingling of segregated cell populations, which in turn exerts varying forces on the cell itself. However, stress distribution within the segregated cluster have not been examined thoroughly. In this study, we investigated the stress distribution during the segregation process of two distinct HACAT+C2C12 cells and MDCK+C2C12 cells. We employed monolayer stress microscopy (MSM) and traction force microscopy (TFM) to map the stresses against each other and the substrate, respectively. Our results showed a clear separation between two cell types where epithelial HACAT gathered as clusters, while mesenchymal C2C12 formed a boundary-like network surrounding the circular clusters. C2C12 cells exhibited notably high traction force and maximum shear stress values along the boundary between two cell types. Our inhibitor studies confirmed the importance of actomyosin contractility in the development of stresses along the boundary, and that of the direction and magnitude of boundary stresses for the maintenance of stable segregation between multiple cell types.