Actin gating of crosstalk between Rho GTPases in cell migration

细胞迁移中 Rho GTP 酶之间串扰的肌动蛋白门控

• 批准号: 10736927
• 负责人: Sean Ryan Collins
• 金额: $ 35.69万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-25 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736927
• 关键词: Actins; Actomyosin; Address; Affect; Arthritis; Atherosclerosis; Autoimmune Diseases; Behavior; Behavior Control; Biochemical; Biological Process; Biosensor; Cell Polarity; Cells; Chemicals; Cues; Cytoskeleton; Defect; Development; Disease; Disseminated Malignant Neoplasm; Drug Targeting; Elements; Environment; Experimental Genetics; F-Actin; Family; Feedback; Fluorescence Resonance Energy Transfer; Glioblastoma; Goals; Guanosine Triphosphate Phosphohydrolases; HL60; Image; Immune; Immune response; Individual; Inflammation; Inflammatory; Leukocytes; Light; Maintenance; Maps; Measurement; Measures; Mediating; Mediator; Microscopy; Modeling; Molecular; Molecular Target; Mutation Analysis; Nature; Neoplasm Metastasis; Optics; Output; Pattern; Play; Process; Proteins; Proteomics; Regulation; Research; Resolution; Role; Signal Pathway; Signal Transduction; Structure; Survival Rate; System; Testing; Tissues; Travel; cancer cell; cell motility; cell type; depolymerization; experimental study; improved outcome; migration; neutrophil; new therapeutic target; novel therapeutic intervention; optogenetics; receptor; recruit; redshift; response; rho; rho GTP-Binding Proteins; scaffold; segregation; tool

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.

项目摘要/摘要 持续的细胞迁移对于免疫复杂，发育和癌症传播至关重要 细胞。这种迁移需要建立和维护稳定的细胞极性，即使在细胞 整合了来自其环境的噪音异质提示。为此，Rho Family GTPases充当了中心 将信号级联和细胞骨架重排成亚细胞结构域组织的枢纽。反馈和 串扰连接被认为是这种形式形成能力的核心。但是，该电路的接线 仍然不完全理解，并且我们对负面调节者如何限制的理解有很大的差距 和单独的空间域。确定迁移白细胞中的这些分子连接将确定 用于治疗炎症的新治疗靶点，将与理解Rho GTPase广泛相关 在许多细胞类型和生物过程中发挥作用。进步的主要障碍是快速的时间表 以及信号系统的固有空间性质。为了应对这些挑战，我们开发了新的 分子工具使我们能够在测量时控制光线的单个关键组件的活动 在活单细胞中具有亚细胞分辨率的第二个成分的响应。我们的初步结果表明 除了充当移动细胞的输出外，不同的肌动蛋白组件与 Rho GTPase串扰的生化布线。我们已经确定了在 RhoA和Cdc42，我们已经确定了蛋白质ARHGAP30为先前未批准的原代调节剂 Cdc42对于白细胞中极化和迁移至关重要。我们假设不同的肌动蛋白 集会充当脚手架，以定位Rho GTPase串扰的调节剂 不同的信号接线以促进稳定的细胞极性。具体而言，我们的目的是1）确定分支肌动蛋白如何 组装调节白细胞细胞中的Cdc42和RhoA活性，2）确定局部肌动蛋白网络如何 结构控制RhoA和Cdc42之间的串扰，3）确定Arhgap30在 串扰和极性信号传导。我们的方法将结合新的工具集，以控制信号传导和 细胞骨架成分，简单地测量肌动蛋白组件和Rho GTPase活性 单细胞。结合使用，我们将使用化学扰动，突变分析和生化 表征分子连接的方法。我们的长期目标是确定如何相互 肌动蛋白和Rho GTPases之间的调节在包括白细胞在内的多种细胞类型中产生了强大的极性 并传播癌细胞。拟议的研究将提高我们对生化如何的基本理解 信号通路会产生和稳定下细胞结构域，以控制细胞迁移等行为。