Regulation of Chemotactic Signaling

趋化信号的调节

• 批准号: 10377388
• 负责人: Miho Iijima
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377388
• 关键词: Arthritis; Asthma; Atherosclerosis; Back; Biochemical; Biological Assay; Biophysical Process; Cell Line; Cell membrane; Cells; Chemicals; Chemotactic Factors; Chemotaxis; Disease; Embryonic Development; Engineering; Fluorescence Resonance Energy Transfer; Immune response; Knock-out; Laboratories; Ligand Binding; Logic; Malignant Neoplasms; Medical; Microscopy; Monomeric GTP-Binding Proteins; Mus; Neoplasm Metastasis; PTEN gene; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Physiological Processes; Proteins; Regulation; Science; Signal Pathway; Signal Transduction; System; Testing; Therapeutic Intervention; Translating; Xenograft Model; cell behavior; cell motility; chronic inflammatory disease; fMet-Leu-Phe receptor; in vivo; interest; live cell imaging; migration; mutant; polarized cell; receptor; reconstitution; rho; single molecule; therapeutic development; tumor; tumorigenesis; wound healing

Summary Chemotactic cell migration underlies embryonic development, wound healing and immune responses. Furthermore, aberrant chemotaxis leads to chronic inflammatory disease and tumor metastasis. My laboratory has long been interested in signaling mechanisms that control cell behaviors in chemotaxis. In a chemical gradient, cells polarize intracellular signaling and migrate toward chemoattractants. Ligand binding to chemoattractant receptors selectively activates two kinases, TORC2 and PI3K, at the front of cells. TORC2 and PI3K are the master kinases that induce cytoskeletal remodeling to extend pseudopods and function immediately downstream of chemotactic receptors. Polarized activation of these two kinases is essential for creating the leading edge of cells that migrate in a chemical gradient. Despite the critical relevance to basic and medical science, an understanding of the spatial and temporal regulation of TORC2 and PI3K remains incomplete. An overarching challenge is to decipher how the TORC2 and PI3K signaling pathways are regulated at the front versus the back of cells in a chemical gradient. To explore mechanisms that regulate the TORC2 pathway, we will take advantage of our recently developed biochemical systems. In these systems, chemoattractant-regulated activation and inhibition of TORC2 are faithfully reconstituted with purified TORC2 and two small GTPases, Rho and Ras. These new assays will identify the biochemical and biophysical mechanisms that create distinct chemotactic signaling at the leading and trailing edges of migrating cells. The mechanistic principle that is determined will be tested and translated in our cellular reconstitution systems using knockout cell lines expressing WT and mutant TORC2 and its regulatory components. Using live-cell imaging with FRET microscopy and single-molecule microscopy, we will place the signaling principle in a spatial and temporal context in migrating cells. For the PI3K pathway, we will analyze proteins that control the localization of the PIP3 phosphatase PTEN to the plasma membrane at the back of cells. The rear localization of PTEN enables PIP3 signaling activation at the leading edge and its inhibition at the trailing edge. We will also determine the function of the identified mechanisms that control PTEN localization in tumorigenesis and metastasis in mouse xenograft models expressing engineered PTEN molecules with altered localization. These studies will elucidate the fundamental logics by which polarization of intracellular signaling is established in cells during chemotactic migration and the physiological importance of this signaling in vivo.

概括 趋化细胞迁移是胚胎发育，伤口愈合和免疫反应的基础。 此外，异常的趋化性导致慢性炎症性疾病和肿瘤转移。我的实验室 长期以来，人们一直对控制趋化性细胞行为的信号传导机制感兴趣。在化学物质中 梯度，细胞极化细胞内信号传导，并向趋化因子迁移。配体结合 化学吸引剂受体在细胞前部有选择地激活两个激酶Torc2和pi3k。 torc2 PI3K是诱导细胞骨架重塑以扩展伪足和功能的主激酶 立即下游趋化受体。这两个激酶的极化激活是必不可少的 用于创建在化学梯度中迁移的细胞的前缘。尽管与 基础和医学，了解TORC2和PI3K的空间和时间调节 仍然不完整。一个总体挑战是破译TORC2和PI3K信号通路如何 在化学梯度中在前面和细胞的背面进行调节。 为了探索调节TORC2途径的机制，我们将利用我们最近开发的 生化系统。在这些系统中，趋化因素调节的激活和TORC2的抑制是 忠实地用纯化的torc2和两个小的GTPases（Rho和Ras）重构。这些新测定将 确定生化和生物物理物理机制，这些机制在领先的 和迁移细胞的尾随边缘。确定的机械原理将进行测试和翻译 在我们的细胞重建系统中，使用表达WT和突变体Torc2及其的基因敲除细胞系 监管组件。使用FRET显微镜和单分子显微镜使用活细胞成像，我们将 将信号原理放在迁移细胞中的空间和时间环境中。对于PI3K途径，我们将 分析控制PIP3磷酸酶PTEN在质膜上定位的蛋白质 细胞的背面。 PTEN的后部定位启用PIP3信号在前沿及其的信号激活 在后缘的抑制。我们还将确定控制的确定机制的功能 在表达工程PTEN的小鼠异种移植模型中的肿瘤发生和转移中的PTEN定位 分子随定位改变。这些研究将阐明基本逻辑 细胞内信号传导的极化是在趋化性迁移期间在细胞中建立的 该信号在体内的生理重要性。