Dynamics of the 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.

   Rho GTPases are important regulators of cytoskeleton dynamics in a variety of biological events including cell division, cell migration, vesicle trafficking and gene expression. Rho GTPases are highly regulated in space and time, key features for their function as molecular switches that transmit environmental cues to intracellular signaling pathways. Moreover, Rho GTPases are themselves regulated by input from the cytoskeleton, coordinating the multiple dynamic responses required by the cell. One biological process that requires precise spatial and temporal coordination of membrane and cytoskeletal components is cell wound repair. Single cell wounds heal by rapidly plugging the plasma membrane disruption with a vesicle patch, and requires the assembly of contractile actomyosin ring and microtubules reorganization. To assess the role of Rho GTPases as regulators of the cell wound repair response, we laser wounded embryos carrying fluorescently-tagged GTPases, then followed their repair in vivo by 3D microscopy. In our single cell model, Rho, Rac (Rac1 and Rac2) and Cdc42 rapidly accumulate around the wound, and segregate into dynamic zones that move inward (basally) as healing progress. 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 in this response. Surprisingly, we find that Rho GTPases utilize specific effectors to mediate their signals: our data shows that different Rho GTPases and their effectors are locally recruited in response to wounding. This complex spatial-temporal array may also involve crosstalk among the different GTPases and their signaling modules. Importantly, by genetic and pharmacological assays we also find that Rho, Rac and Cdc42 are required for proper wound repair, and each of them make specific contributions to the assembly and organization of the actomyosin array.