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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要持续的细胞迁移是免疫反应、癌症发展和传播的基础这种迁移需要建立和维持稳定的细胞极性，即使是在细胞存在时也是如此。整合来自环境的嘈杂异质线索为了实现这一目标，Rho 家族 GTPases 发挥着核心作用。将信号级联和细胞骨架重排组织成亚细胞域的中枢。串扰连接被认为是这种图案形成能力的核心，但是该电路的布线。仍然不完全被理解，并且我们对负监管机构如何限制的理解存在重大差距确定迁移白细胞中的这些分子连接将识别出不同的空间域。治疗炎症的新治疗靶点，与了解 Rho GTPase 具有广泛的相关性在许多细胞类型和生物过程中发挥作用的主要障碍是快速的时间尺度。为了应对这些挑战，我们开发了新的信号系统。分子工具使我们能够在测量光的同时控制各个关键成分的活性我们的初步结果表明，第二个成分在活单细胞中具有亚细胞分辨率。除了充当移动细胞的输出之外，不同的肌动蛋白组件还密切参与 Rho GTPase 串扰的生化布线我们已经确定了 Rho GTPase 串扰之间的“肌动蛋白门控”串扰连接。 RhoA 和 Cdc42，并且我们已经确定蛋白质 Arhgap30 是以前未被重视的主要调节因子 Cdc42 对白细胞的极化和迁移至关重要。组件充当支架来定位 Rho GTPase 串扰的调节器，从而创建亚细胞区域具体来说，我们的目标是 1) 确定肌动蛋白的分支方式。组装调节白细胞中的 Cdc42 和 RhoA 活性，2) 决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 在我们的方法将结合用于信号传输和极性信号光学控制的新工具集。细胞骨架成分，同时测量肌动蛋白组装和 Rho GTPase 活性结合起来，我们将使用化学扰动、突变分析和生化分析。我们的长期目标是确定分子连接的相互关系。肌动蛋白和 Rho GTP 酶之间的调节在多种细胞类型（包括白细胞）中产生强大的极性拟议的研究将增进我们对生物化学如何传播的基本了解。信号通路产生并稳定亚细胞结构域以控制细胞迁移等行为。