Calcium oscillations in wounded fibroblast monolayers are spatially regulated through substrate mechanics

摘要

The maintenance of tissue integrity is essential for the life of multicellular organisms. Healing of a skin wound is a paradigm for how various cell types localize and repair tissue perturbations in an orchestrated fashion. To investigate biophysical mechanisms associated with wound localization, we focus on a model system consisting of a fibroblast monolayer on an elastic substrate. We find that the creation of an edge in the monolayer causes cytosolic calcium oscillations throughout the monolayer. The oscillation frequency increases with cell density, which shows that wound-induced calcium oscillations occur collectively. Inhibition of myosin II reduces the number of oscillating cells, demonstrating a coupling between actomyosin activity and calcium response. The spatial distribution of oscillating cells depends on the stiffness of the substrate. For soft substrates with a Young's modulus E similar to 360 Pa, oscillations occur on average within 0.2 mm distance from the wound edge. Increasing substrate stiffness leads to an average localization of oscillations away from the edge (up to similar to 0.6 mm). In addition, we use traction force microscopy to determine stresses between cells and substrate. We find that an increase of substrate rigidity leads to a higher traction magnitude. For E < similar to 2 kPa, the traction magnitude is strongly concentrated at the monolayer edge, while for E > similar to 8 kPa, traction magnitude is on average almost uniform beneath the monolayer. Thus, the spatial occurrence of calcium oscillations correlates with the cell-substrate traction. Overall, the experiments with fibroblasts demonstrate a collective, chemomechanical localization mechanism at the edge of a wound with a potential physiological role.