Macromolecular Engineering

摘要 所有项目都提议使用能够进行操纵和审讯的新型工程试剂 具有精确的空间和时间控制的个体信号通路。如每个项目中所述， Core C提供的试剂将用于定义调节肺恢复的过程 内皮屏障。开发和优化这些工具需要付出巨大的努力，因为 在分子工程核心C中描述。核心C的中心功能将是1)开发 针对单个项目中的每个特定问题定制的分子工具，2)评估新的 试剂盒及其在人和小鼠肺微血管中的应用 内皮细胞，以及3)在工具的应用、故障排除和 对结果进行分析。其目的是简化新技术在该计划中的应用 项目并允许与会者集中讨论需要这些试剂的拟议问题。这个 Core C将开发和使用的特定试剂包括：1)新的控制工具 每个项目确定的蛋白质的定位和相互作用；2)活性调节试剂 在活细胞中选择蛋白质，并在特定的复合体中定向激活这些蛋白质 亚细胞定位；以及3)光介导的小细胞时空调节试剂 活细胞中的GTP酶rac1和CDc42以及其他GTP酶的需要。核心C还将生成和 优化在原代内皮细胞和动物模型中表达工程蛋白的试剂， 并开发用于操纵和询问蛋白质相互作用和细胞信号的新试剂 视需要而定。开发的工具将能够操纵单独的信号通路和 评估它们在调节内皮屏障恢复和恢复完整性中的作用 粘着的结合处。

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.