Macromolecular Engineering

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Abstract All Projects propose to employ novel engineered reagents that enable manipulation and interrogation of individual signaling pathways with precise spatial and temporal control. As described in each Project, reagents provided by Core C will be used to define the processes that regulate the recovery of the lung endothelial barrier. Development and optimization of these tools will require significant effort as described in the Molecular Engineering Core C. The central functions of Core C will be 1) to develop molecular tools customized for each specific question in individual Projects, 2) to evaluate the new reagents and establish protocols for their application in primary human and mouse lung microvessel endothelial cells, and 3) to provide assistance with the application of the tools, troubleshooting, and analysis of the results. The intent will be to simplify application of new technologies in the Program Project and allow participants to focus on the proposed questions requiring these reagents. The specific reagents that will be developed and employed by Core C include: 1) new tools to control localization and interaction of proteins identified by each Project; 2) reagents for regulation of activity of selected proteins in living cells, and targeted activation of these proteins in specific complexes and subcellular locations; and 3) reagents for light-mediated spatio-temporal regulation of the small GTPases Rac1 and Cdc42 and other GTPases in living cells as needed. Core C will also generate and optimize reagents for expression of engineered proteins in primary endothelial cells and animal models, and develop new reagents for manipulation and interrogation of protein interactions and cell signaling as needed. The tools developed will enable manipulation of individual signaling pathways and assessment of their roles in the regulation of endothelial barrier recovery and restoration of the integrity of adherens junctions.