Spatio-temporal dynamics of GEF-GTPase networks

描述(申请人提供)：Rho家族GTP酶是普遍存在的分子开关，控制着非常不同的细胞过程。它们被鸟嘌呤核苷酸交换因子(GEF)激活，GEF的数量大约是GTP酶本身的5倍，并整合了许多控制GTP酶功能的细胞输入。GEFS和GTP酶形成复杂的网络，这些网络是为特定目的而临时和局部地构成的。生化、遗传、分子和结构分析已经揭开了许多关于这些至关重要的途径的谜团，但最重要的功能特性，即它们的时空调节，只有在完整细胞的背景下才能完全理解。该PPG将具有不同专业知识的团队成员聚集在一起，开发创新技术，从而能够研究活体中的全球环境基金/GTPase网络，从成像数据中提取网络架构和信号动力学的计算工具，以及对严重依赖于全球环境基金/GTPase动态的细胞行为的深入知识：(项目1-HAHN)将根据针对不同全球环境基金结构类别的设计提供全球环境基金生物传感器。在与全球环境基金结构专家Sondek的合作中，不同的生物传感器设计将报告特定上游输入对全球环境基金的激活，以及内源性GEF的激活。(项目2--Danuser)将开发同时对任何一对全球环境基金和GTP酶的活性进行成像和/或光学操纵的能力，以便高分辨率地研究全球环境基金/GTP酶的时空协调。新的计算工具将结合不同实验的数据来对大型网络进行建模，并从成像数据中提取网络架构和信号动力学。这些方法将在复杂的全球环境基金-GTPase反馈相互作用的研究中进行测试。(项目3-霍尔)：这个以生物学为重点的项目将把我们的工作扩展到多细胞系统。我们将重点研究细胞-细胞连接和隐形片层脂蛋白中的全球环境基金激活，并确定调节集体迁移的全球环境基金。(项目4--布里奇)将探讨全球环境基金/GTP酶网络在机械转导中的作用，探索关于RhoA信号在细胞基质和细胞-细胞黏附开始突起期间以及在细胞核中的机械调节的新发现。

DESCRIPTION (provided by applicant): Rho family GTPases is ubiquitous molecular switches that control extraordinarily diverse cellular processes. They are activated by guanine nucleotide exchange factors (GEFs) that are roughly 5-fold more numerous than the GTPases themselves and integrate the many cellular inputs controlling GTPase function. GEFs and GTPases form complex networks that are constituted transiently and locally for specific purposes. Biochemical, genetic, molecular, and structural analyses have unraveled a great deal about these critically important pathways, but the most important functional property, their spatio-temporal regulation, can only be fully understood in the context of intact cells. This PPG brings together team members with diverse expertise to develop innovative technologies enabling the study of GEF/GTPase networks in vivo, computational tools to extract network architecture and signaling kinetics from imaging data, and in-depth knowledge of cell behaviors critically dependent on GEF/GTPase dynamics: (Project 1- Hahn) will deliver GEF biosensors based on designs addressing different GEF structural classes. In a collaborative effort with Sondek, expert in GEF structure, different biosensor designs will report GEF activation by specific upstream inputs, and activation of endogenous GEFs. (Project 2- Danuser) will develop the ability to simultaneously image and/or photomanipulate the activity of any pair of GEFs and GTPases, for high resolution studies of GEF/GTPase spatio-temporal coordination. New computational tools will combine data from different experiments to model large networks, and to extract network architecture and signaling kinetics from imaging data. These methods will be tested in studies of complex GEF-GTPase feedback interactions. (Project 3- Hall): This biologically focused project will extend our work to multicellular systems. We will focus on GEF activation in cell-cell junctions and cryptic lamellipodia, and identify GEFs regulating collective migration. (Project 4- Burridge) will address the role of GEF/GTPase netvvorks in mechanotransduction, exploring novel findings regarding the mechanical regulation of RhoA signaling at cell-matrix and cell-cell adhesions during initiation of protrusions, and in the nucleus.