Role of Calcium and Rho family small GTPases in Single Cell Wound Repair. Maria Teresa Abreu-Blanco, Susan M Parkhurst. Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

   Cells and tissues are constantly exposed to mechanical and physical stresses, and their ability to respond to damage is critical for their survival. In particular, single cells must quickly repair their wounds to avoid cell death due to loss of cytoplasm and influx of ions. In Drosophila, single cell wound repair is mediated by specific spatial and temporal responses: plasma membrane is recruited as vesicles and from the wound border, an actomyosin ring is assembled serving as the contractile force driving closure, and the ring and plasma membrane are linked by E-Cadherin. We find that actin, Myosin II, Microtubules and E-Cadherin are all required for single cell wound repair. To the date, the only known signaling molecule that can trigger cell wound repair is calcium. In Xenopus oocytes the influx of calcium from the environment triggers membrane recruitment to the wound and its fusion with the plasma membrane. In the Drosophila embryo, we observed a wave of calcium around the wound area as soon as 10s post-wounding. We are currently investigating the role of this calcium wave in wound repair, and which downstream molecules are mediating this signal. Rho GTPases are well known cytoskeleton modulators and have been involved in coordinating multiple dynamic responses required by the cell. In our single cell model, Rho, Rac and Cdc42 rapidly accumulate around the wound, and segregate into dynamic zones. Importantly, genetic and pharmacological assays show that Rho, Rac and Cdc42 are required for wound repair, and each of them makes specific contributions to the assembly and organization of the actomyosin array. We also developed biosensor probes for each GTPase, using the Rho binding domains of different downstream effectors, to determine the spatial and temporal dynamics of active GTPases during the repair process. We find that Rho GTPases utilize specific effectors to mediate their signals. Significantly, we also observed crosstalk between the different GTPases and their signaling modules and the cytoskeleton.