Actin gating of crosstalk between Rho GTPases in cell migration

项目总结/摘要 持续的细胞迁移是免疫反应、发展和癌症传播的基础 细胞这种迁移需要建立和维持稳定的细胞极性，即使细胞 整合来自环境的嘈杂的异质线索。为了实现这一点，Rho家族GTP酶作为核心 组织信号级联和细胞骨架重排进入亚细胞结构域的枢纽。反馈和 串扰连接被认为是这种图案形成能力的核心。然而，这个电路的布线 仍然没有完全理解，我们对负调节器如何限制 分离的空间域。确定这些迁移白细胞中的分子连接， 这是治疗炎症的新的治疗靶点，并将与理解Rho GT3广泛相关 在许多细胞类型和生物过程中发挥作用。进展的主要障碍是时间表过快 以及信号系统固有的空间特性。为了应对这些挑战，我们开发了新的 分子工具，使我们能够控制活动的个别关键组成部分与光，同时测量 在活的单细胞中具有亚细胞分辨率的第二组分的响应。我们的初步结果表明 除了作为输出来移动细胞外，不同的肌动蛋白组件密切参与了细胞的运动。 Rho GT3串扰的生化布线。我们已经确定了一个“肌动蛋白门控”串扰连接之间 RhoA和Cdc 42，我们已经确定了蛋白质Arhgap 30作为一个以前不受重视的主要调节因子 Cdc 42是白细胞极化和迁移的关键。我们假设不同肌动蛋白 组装作为支架定位Rho GT3串扰的调节器，产生亚细胞区， 独特的信号线，以促进稳定的细胞极性。具体来说，我们的目标是1）确定分支肌动蛋白 组装调节白细胞中Cdc 42和RhoA活性，2）确定局部肌动蛋白网络如何 结构控制RhoA和Cdc 42之间的串扰，以及3）确定Arhgap 30在RhoA和Cdc 42中的调节和作用。 串扰和极性信令。我们的方法将结合联合收割机新的工具集，用于光控制信令， 细胞骨架成分，同时测量肌动蛋白组装和Rho GT3活性， 单细胞结合起来，我们将使用化学扰动，突变分析和生化分析。 表征分子连接的方法。我们的长期目标是确定 肌动蛋白和Rho GTP酶之间的调节在多种细胞类型中产生了强大的极性，包括白细胞 和扩散癌细胞。这项拟议中的研究将推进我们对生物化学如何 信号通路产生并稳定亚细胞结构域以控制诸如细胞迁移的行为。

PROJECT SUMMARY/ABSTRACT Persistent cell migration is fundamental for immune responses, development, and the dissemination of cancer cells. This migration requires the establishment and maintenance of stable cell polarity, even while a cell integrates noisy heterogeneous cues from its environment. To achieve this, Rho family GTPases act as central hubs that organize signaling cascades and cytoskeletal rearrangements into subcellular domains. Feedback and crosstalk connections are thought to be central to this pattern-forming ability. However, the wiring of this circuit is still incompletely understood, and there are major gaps in our understanding of how negative regulators limit and separate spatial domains. Determining these molecular connections in migrating leukocytes would identify new therapeutic targets for treating inflammation and would be broadly relevant for understanding Rho GTPase function in many cell types and biological processes. Major obstacles to progress have been the fast timescale and inherently spatial nature of the signaling system. To address these challenges, we have developed new molecular tools that allow us to control the activity of individual key components with light while measuring the response of a second component with subcellular resolution in live single cells. Our preliminary results indicate that in addition to acting as outputs to move the cell, different actin assemblies are intimately involved in the biochemical wiring of Rho GTPase crosstalk. We have identified an “actin-gated” crosstalk connection between RhoA and Cdc42, and we have identified the protein Arhgap30 as a previously unappreciated primary regulator of Cdc42 that is critical for polarization and migration in leukocytes. We hypothesize that different actin assemblies act as scaffolds to localize regulators of Rho GTPase crosstalk, creating subcellular zones with distinct signal wiring to promote stable cell polarity. Specifically, we aim to 1) determine how branched actin assembly regulates Cdc42 and RhoA activities in leukocyte cells, 2) determine how the local actin network structure controls crosstalk between RhoA and Cdc42, and 3) determine the regulation and role of Arhgap30 in crosstalk and polarity signaling. Our approach will combine new tool sets for optical control of signaling and cytoskeletal components with simultaneous measurement of actin assemblies and Rho GTPase activities in single cells. In combination, we will use chemical perturbations, mutational analysis, and biochemical approaches to characterize molecular connections. Our long-term goals are to determine how reciprocal regulation between actin and Rho GTPases creates robust polarity in multiple cell types, including leukocytes and disseminating cancer cells. The proposed research will advance our basic understanding of how biochemical signaling pathways both generate and stabilize subcellular domains to control behaviors such as cell migration.