Project Summary The parent grant investigates wound-induced syncytia in an epithelium in vivo – from how syncytia form to how they participate in closing wounds to how they are eliminated after a wound closes. We specifically analyze syncytia around laser wounds, and the laser ablation is conducted on a confocal microscope, allowing us to image before, during, and after ablation with very high temporal resolution – as fast as a 30-millisecond frame rate. Our current Nd:YAG laser generates a gradient of damage across the wound bed, with the central area comprised of cell lysis, and a peripheral area of plasma membrane damage from which cells can recover. We have evidence that syncytia arise from the fusion of cells within the region of plasma membrane damage: wound-induced epithelial fusions localize to this area, and cells begin to share their cytoplasmic contents with their neighbors within 500 ms after wounding, suggesting that the damaged membranes are fusogenic. What we need, however, is to administer laser wounds in such a way that they do not generate plasma membrane damage; if we can do this, we predict that no syncytia would form. Our current laser is not suited to this task, so we propose to purchase a compact fiber laser with sub-100-fs pulses, which would deliver uniform damage across the wound area in a fraction of a second. This equipment, not available anywhere on campus, would allow us to complete the aims of the parent proposal.