G-protein Coupled Receptor Mediated Chemoattractant Sensing and Phagocytosis

G 蛋白偶联受体介导的趋化剂感应和吞噬作用

• 批准号: 7964478
• 负责人: Tian Jin
• 金额: $ 80万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 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/7964478
• 关键词: Actins; Back; Biological Models; Caenorhabditis elegans; Cells; Chemotactic Factors; Chemotaxis; Complex; Computer Simulation; Confocal Microscopy; Coupled; Cyclic AMP; Cytoskeleton; DNA Sequence Rearrangement; DOCK1 protein; Defect; Dictyostelium discoideum; E protein; Endosomes; Event; Foundations; G Protein-Coupled Receptor Genes; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Protein beta Subunits; GTP-Binding Proteins; Genes; Goals; Homologous Gene; Host Defense; Human body; Imagery; Integrins; Life; Lysosomes; Mediating; Membrane; Microfilaments; Molecular; Nutrient; Phagocytes; Phagocytosis; Phagosomes; Process; Protein Family; Protein Tyrosine Kinase; Proteins; Receptor Protein-Tyrosine Kinases; Reporting; Role; Signal Transduction; Signaling Protein; Spatial Distribution; Surface; System; Vesicle; Work; base; cell motility; chemokine receptor; combat; macrophage; microbial; model development; novel; particle; pathogen; polymerization; prevent; receptor; research study; response

We are investigating the following questions: 1. how a GPCR chemosensing network regulates the polarized reorganization of the actin cytoskeleton required for protrusion of the cell's front and retraction of its back during chemotaxis. 2. What are the molecular mechanisms underlying phagosome maturation process during phagocytosis? 1. In recent years, the Elmo (Engulfment and Motility) protein family has been implicated in actin cytoskeleton reorganization during both phagocytosis and chemotaxis. ELMO was first discovered as an essential component involved in engulfment of dead cells in C. elegans. DOCK180 and ELMO form a complex that functions as a bipartite GEF to optimally activate Rac and promote actin cytoskeleton rearrangement. DOCK-ELMO complexes also receive signals from GPCRs, integrins and tyrosine kinases to activate Rac, leading to cell migration. However, how GPCRs and other receptors activate DOCK-ELMO has not been determined in any system. As both DOCK and ELMO are evolutionarily conserved, we have been using D. discoideum, as a model system to study the functions of ELMO (A-F) proteins in GPCR-mediated signaling network leading to chemotaxis. We have identified six Elmo homologs in D. discoideum, and have reported that ElmoA functions to maintain cell polarization by preventing excessive actin polymerization around the cell periphery during phagocytosis and chemotaxis. We have been investigating the roles of ELMO-E in chemotaxis. We performed gene disruption, replacement, and over-expression experiments. We found that cells lacking ELMO-E display chemotaxis defect. Visualization of YFP tagged ELMO-E by confocal microscopy revealed ELMO-E translocates to regions of newly forming actin filaments, colocalizing at the leading edge of dominant pseudopod in chemotaxing cells. ELMO-E transiently translocates to membrane under cAMP stimulation by the facility of actin polymerization. This translocation is PI3K independent. More interestingly, we found that ELMO-E interacts with G protein beta subunit and may associate with Rac family proteins as well. Taken together, we propose that ELMO-E protein may be the missing connection between the GPCR/G-protein, the chemoattractant sensing machinery, and the actin cytoskeleton, the machinery of cell movement. 2. Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered from phagosomes to lysosomes for degradation, but the molecular mechanism regulating phagosome maturation remains unclear. Using D. discoideum as a model system, we plan to reveal important components involved in phagosome maturation. We have identified 3 novel vesicle-associated receptor tyrosine kinases, VSK1-3, in D. discoideum. Our previous study suggests that localized VSK3 tyrosine kinase signaling on the surface of endosome/lysosomes represents a new control mechanism for phagosome maturation. We are identifying targets of VSK 2 and 3. This study will provide a foundation for understanding the molecular mechanism of VSK signaling that regulate phagosome maturation.

我们正在研究以下问题： 1. GPCR 化学传感网络如何调节趋化过程中细胞正面突出和背面缩回所需的肌动蛋白细胞骨架的极化重组。 2. 吞噬过程中吞噬体成熟过程的分子机制是什么？ 1. 近年来，Elmo（吞噬和运动）蛋白家族在吞噬作用和趋化作用过程中参与了肌动蛋白细胞骨架的重组。 ELMO 最初被发现是参与线虫死亡细胞吞噬的重要成分。 DOCK180 和 ELMO 形成复合物，作为二分 GEF 发挥作用，以最佳方式激活 Rac 并促进肌动蛋白细胞骨架重排。 DOCK-ELMO 复合物还接收来自 GPCR、整合素和酪氨酸激酶的信号以激活 Rac，从而导致细胞迁移。然而，GPCR 和其他受体如何激活 DOCK-ELMO 在任何系统中尚未确定。由于 DOCK 和 ELMO 在进化上都是保守的，因此我们一直使用 D. discoideum 作为模型系统来研究 ELMO (A-F) 蛋白在 GPCR 介导的趋化信号网络中的功能。 我们在 D. discoideum 中鉴定了 6 个 Elmo 同源物，并报道 ElmoA 通过防止吞噬和趋化过程中细胞周围肌动蛋白过度聚合来维持细胞极化。 我们一直在研究 ELMO-E 在趋化性中的作用。我们进行了基因破坏、替换和过度表达实验。我们发现缺乏 ELMO-E 的细胞表现出趋化性缺陷。 通过共聚焦显微镜对 YFP 标记的 ELMO-E 进行可视化显示，ELMO-E 易位到新形成的肌动蛋白丝区域，共定位于趋化细胞中优势伪足的前缘。 ELMO-E 在 cAMP 刺激下通过肌动蛋白聚合设施瞬时易位至膜。 该易位与 PI3K 无关。更有趣的是，我们发现 ELMO-E 与 G 蛋白 β 亚基相互作用，也可能与 Rac 家族蛋白相关。 综上所述，我们认为 ELMO-E 蛋白可能是 GPCR/G 蛋白（趋化剂传感机制）和肌动蛋白细胞骨架（细胞运动机制）之间缺失的连接。 2. 吞噬作用对于宿主防御微生物病原体和盘基网柄菌获取营养至关重要。吞噬颗粒从吞噬体递送至溶酶体进行降解，但调节吞噬体成熟的分子机制仍不清楚。使用 D. discoideum 作为模型系统，我们计划揭示参与吞噬体成熟的重要组成部分。我们在盘状 D. 中鉴定出 3 种新型囊泡相关受体酪氨酸激酶 VSK1-3。我们之前的研究表明，内体/溶酶体表面的局部 VSK3 酪氨酸激酶信号传导代表了吞噬体成熟的新控制机制。我们正在确定 VSK 2 和 3 的靶标。这项研究将为理解调节吞噬体成熟的 VSK 信号传导的分子机制奠定基础。