| The boundary between life and death for cells is razor-thin. Less than ten nanometers of a flexible, selectively permeable lipid bilayer separate the harsh extracellular environment from the cytoplasm. Although assaults to these barriers are common, damage can be limited by evolutionarily-conserved mechanisms employed to repair plasma membrane defects. The wound response is shaped by a series of overlapping signaling gradients including calcium and membrane lipids. These gradients are used to construct complex patterns such as concentric zones of active Rho and Cdc42 which, in turn, drive the formation and ingression of corresponding rings of myosin-2 and F-actin, respectively. Organisms use many of these same patterns to close multicellular wounds. My work endeavors to identify and characterize factors that generate and refine the patterns seen at wounds. In Chapter 2, I continued a candidate screen initiated by Vaughan et al. (2011) in search of regulators of Rho GTPase activity zones at wounds in Xenopus laevis oocytes. I examined the roles of two Rho GTPase inactivators, RhoGAP1 and RhoGAP8. I found that they localize between the zones of active Rho and Cdc42 and are responsible for modulating Rho zone width. In Chapter 3, I expand the characterization of RhoGAP1 and RhoGAP8 to determine their function in the intact epithelia of Xenopus embryos. I found that RhoGAP1 and RhoGAP8 both localize to multicellular wounds. Knockdown of RhoGAP1 causes both defects during gastrulation and increased cellular F-actin. In Chapter 4, a combination of traditional cell-labelling approaches and recent advances in confocal microscopy were used to study the dynamics of calcium, various membranous compartments, and selected membrane-associated proteins immediately after wounding at higher spatiotemporal resolution than previously possible. Membrane compartments of various sizes were observed undergoing complex fusion events to shape the lipidic environment around the wound. Combined, the results of this work add to a growing list of processes and participants needed to repair wounded cells. More importantly, these findings demonstrate additional mechanisms for shaping intracellular patterns generated during wound repair and may serve as a proxy for other forms of single-cell pattern formation. |
