Model wounds in a model organism: tracking subcellular damage and movements around laser-generated wounds in Drosophila pupae*

A major challenge in biology and medicine is understanding the mechanisms of epithelial wound healing, i.e., identifying how an epithelial tissue repairs a wound through coordinated and patterned changes in cellular behavior. A key question is how the patterns of cell behavior relate to the patterns of cellular damage around a wound. To investigate this relationship, we make laser wounds in the notal epithelium of Drosophila pupae. Laser ablation makes a controlled and reproducible pattern of cellular damage via both plasma generation at the focus and a rapidly expanding and collapsing cavitation bubble, which applies shear stress that damages the plasma membrane of surrounding cells. The subcellular location of this damage can be tracked using live-organism confocal fluorescent microscopy to track the patterns of rapid calcium influx on millisecond time scales. Similar time-lapse microscopy with fluorescently labeled adherens junctions allows us to track how cells move over the first few seconds after wounding – due to a combination of forces from the wounding process itself and the subsequent opening of the wound driven by epithelial tension. Combining the patterns of fast wound-induced calcium influx and movement, we can relate shear-stress patterns to damage around the wound and beyond to the pattern of cell behaviors observed on longer time scales.