When an epithelial tissue is wounded, the cells undergo a set of coordinated behaviors to close the wound and repair the tissue. Cells across the epithelium participate in this response, not just the cells bordering the wounds. We want to know how cells, including those away from the wound site, get information that a wound has occurred. The earliest observed wound response is a calcium wave, previously reported to originate in cells on the wound margin and expand outward several cells in diameter. This calcium wave is highly conserved and is important for wound healing. Normally, calcium concentrations inside cells are maintained at low levels by pumping calcium out of the cell or into the ER. When the cytoplasmic concentrations rise, calcium can act as a second messenger and can affect multiple signaling pathways. However, the mechanism of wave initiation remains unknown. Based on the calcium wave kinetics we have observed, we hypothesize that the calcium wave upon wounding is a result of changes in tissue mechanics.
We have analyzed the kinetics of the calcium wave using in vivo live imaging of the Drosophila pupae notum, an epithelial monolayer of diploid cells that expresses the GFP-based GCaMP calcium reporter. Our data reveals two distinct stages of calcium dynamics upon wounding; an initial release and stochastic flares. During the initial release, calcium flows simultaneously into multiple cells (at varying distances from the wound margin), only milliseconds after wounding. The second stage of calcium release occurs ~30-60 seconds after wounding and is characterized by random flashes of calcium that propagate around the wound for up to 30 minutes. These flashes are consistent with calcium induced calcium release.
The spread of the initial release from cells proximal to the wound toward cells distal to the wound is on the order of 1-10 µm/ms. Preliminary analysis suggests that diffusion cannot account for such a rate of spread. Thus we are testing the role of mechanotransduction in wound detection. We will use genetic tools to manipulate tissue mechanics and then assess how the calcium wave is affected by the mechanical perturbations.