The mechanisms underlying the GPCR-mediated chemotaxis in D. discoideum

D. discoideum GPCR 介导的趋化机制

基本信息

批准号：
9566620
负责人：
Tian Jin
金额：
$ 61.81万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9566620
关键词：
Actins Amoeba genus Animal Model Bacteria Bacterial Infections Binding Biochemical Biological Models Blood Circulation Cells Chemicals Chemotactic Factors Chemotaxis Computer Assisted Coupled Cyclic AMP Cyclic AMP Receptors Cytoskeleton Cytosol Data Set Defect Dictyostelium discoideum Dissociation Escherichia coli Eukaryotic Cell F-Actin Folic Acid Food G-Protein-Coupled Receptors G-substrate GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Glutaminase Goals Heterotrimeric GTP-Binding Proteins Hormones Human Human body Immune Immunology Impairment Infection Investigation Knock-out Light Mediating Membrane Modeling Molecular Movement Natural Immunity Neurotransmitters Organism Phagocytes Phagocytosis Physiological Process Proteins Recruitment Activity Signal Pathway Signal Transduction Signal Transduction Pathway Site Source Stimulus Surface Techniques base cell motility chemokine directional cell extracellular fMet-Leu-Phe receptor fighting fluorescence imaging genome database imaging modality immune phagocytosis macrophage migration neutrophil novel particle pathogen phosphoproteomics polymerization receptor-mediated signaling response spatiotemporal

项目摘要

1: Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in Dictyostelium discoideum. We discover a novel negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells, is activated by and is essential for GPCR-mediated Ras adaptation. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation recruits C2GAP1 from cytosol and retains it on the membrane to locally inhibit Ras signaling. The altered Ras activation results in impaired gradient sensing and excessive polymerization of F-actin in c2gap1 knockout (c2gap1-) cells, leading to chemotaxis defects. Remarkably, c2gap1- cells display altered cell response, impaired directional sensing, and chemotaxis defects in a chemoattractant concentration-dependent fashion. Thus, we have uncovered a novel inhibitory mechanism required for the adaptation and long-range chemotax.(Xu et al., in revision). 2: Human phagocytes, including neutrophils and macrophage, are an essential part of innate immunity. Upon bacterial infection, neutrophils leaves circulation and migration to infection sites to fight pathogens. They seek bacteria by detecting chemoattractants generated from the bacterial infection site and chase them via chemotaxis. Once reaching the site, they bind and ingest bacteria by recognizing signals form the bacterial surface via phagocytosis. Dictyostelium discoideum amoeba are professional phagocytes that track down bacteria by chemotaxis and capture and ingest them as food through phagocytosis. Studies in the simple model organism of D. discoideum have made tremendous contribution to our current understanding of molecular mechanisms underlying chemotaxis and phagocytosis of phagocytes. Recently, we discovered that D. discoideum amoeba use a chemoattractant GPCR fAR1 to detect folic acid released from bacteria for both chemotaxis to catch bacteria and phagocytosis to ingest them (Pan et al, 2016). This finding suggest to us that a chemoattractant GPCR-mediated signaling network controls reorganization of the actin cytoskeleton for both chemotaxis and phagocytosis, which represents a paradigm-shifting new concept in immunology. Thus, investigation of molecular components involved in chemotaxis and (or) phagocytosis in D. discoideum will continually shed light on the molecular mechanisms controlling migration of immune cells and as well as phagocytosis by immune cells to eliminate bacterial pathogens from human body. Recently, we discovered that formyl-peptide receptors (fPR GPCR) coupled with heterotrimeric Gi proteins mediate chemotaxis as well as phagocytosis of fMLP-coated particles and E. coli. Our studies revealed an evolutionarily conserved mechanism that directs professional phagocytes migrating toward bacteria via chemotaxis and promotes them to engulf bacterial via surface phagocytosis as an essential part of innate immunity.

图 1：真核细胞在各种趋化剂浓度梯度下趋化，因此需要终止细胞反应的抑制过程以达到适应，同时保持对较高浓度刺激的敏感性。然而，抑制过程背后的分子机制仍然知之甚少。在这里，我们揭示了盘基网柄菌趋化性 GPCR 介导的信号网络中局部控制的抑制过程。我们发现了 Ras 信号传导的新型负调节因子 C2GAP1，它位于趋化细胞的前缘，被 GPCR 介导的 Ras 适应激活，并且对于 GPCR 介导的 Ras 适应至关重要。我们发现，C2 和 GAP 结构域都是 C2GAP1 膜靶向所必需的，并且 GPCR 触发的 Ras 激活会从细胞质中招募 C2GAP1 并将其保留在膜上以局部抑制 Ras 信号传导。 Ras 激活的改变会导致 c2gap1 敲除 (c2gap1-) 细胞中的梯度感应受损和 F-肌动蛋白过度聚合，从而导致趋化性缺陷。值得注意的是，c2gap1-细胞以趋化剂浓度依赖性方式表现出细胞反应改变、方向感应受损和趋化性缺陷。因此，我们发现了适应和长程趋化所需的一种新的抑制机制。（Xu等人，修订中）。 2：人类吞噬细胞，包括中性粒细胞和巨噬细胞，是先天免疫的重要组成部分。细菌感染后，中性粒细胞离开循环并迁移到感染部位以对抗病原体。它们通过检测细菌感染部位产生的化学引诱剂来寻找细菌，并通过趋化性追逐它们。一旦到达该位点，它们就会通过吞噬作用识别细菌表面的信号来结合并摄取细菌。盘基网柄菌阿米巴原虫是专业的吞噬细胞，通过趋化作用追踪细菌，并通过吞噬作用捕获并摄取它们作为食物。对盘状 D. discoideum 简单模型生物的研究为我们目前对吞噬细胞趋化和吞噬作用的分子机制的理解做出了巨大贡献。最近，我们发现盘状变形虫使用化学引诱剂 GPCR fAR1 来检测细菌释放的叶酸，以实现趋化捕获细菌和吞噬细菌的作用 (Pan et al, 2016)。这一发现向我们表明，趋化剂 GPCR 介导的信号网络控制肌动蛋白细胞骨架的重组，以实现趋化和吞噬作用，这代表了免疫学中范式转变的新概念。因此，对盘状 D. discoideum 中参与趋化和（或）吞噬作用的分子成分的研究将不断揭示控制免疫细胞迁移和免疫细胞吞噬作用以消除人体内细菌病原体的分子机制。最近，我们发现甲酰基肽受体 (fPR GPCR) 与异三聚体 Gi 蛋白偶联可介导 fMLP 包被颗粒和大肠杆菌的趋化性以及吞噬作用。我们的研究揭示了一种进化上保守的机制，引导专业吞噬细胞通过趋化性向细菌迁移，并促进它们通过表面吞噬作用吞噬细菌，这是先天免疫的重要组成部分。