Regulation of Chemotactic Signaling

趋化信号的调节

• 批准号: 10798693
• 负责人: Miho Iijima
• 金额: $ 1.34万
• 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/10798693
• 关键词: Arthritis; Asthma; Atherosclerosis; Back; Biochemical; Biological Assay; Biophysical Process; Cell Line; Cell membrane; Cells; Chemicals; Chemotactic Factors; Chemotaxis; Cytoskeleton; 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; PIK3CG gene; 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 长期以来，实验室一直对控制趋化性细胞行为的信号传导机制感兴趣。在 一种化学梯度，细胞极化细胞内信号传导，并朝向趋化剂迁移。配体 与趋化受体结合选择性地激活两个激酶，即Torc2和PI3K，在 细胞。 TORC2和PI3K是诱导细胞骨架重塑延伸的主激酶 伪足和功能立即在趋化受体的下游。偏振活化 这两个激酶对于创建在化学物质中迁移的细胞的前缘至关重要 坡度。尽管与基础和医学有关键的相关性，但对空间和 TORC2和PI3K的时间调节仍然不完整。一个总体挑战是破译 TORC2和PI3K信号通路如何在正面与细胞背面调节 化学梯度。 为了探索调节TORC2途径的机制，我们将利用我们最近的优势 开发的生化系统。在这些系统中，趋化因素调节的激活和抑制 TORC2忠实地通过纯化的TORC2和两个小的GTPases（Rho和Ras）重组。这些 新测定将确定生化和生物物理机制，这些机制会产生独特的趋化性 在迁移细胞的前沿和后尾的信号传导。确定的机械原理 将使用表达敲除细胞系在我们的蜂窝重构系统中测试和翻译 WT和突变的Torc2及其调节组件。使用FRET显微镜使用活细胞成像 和单分子显微镜，我们将将信号原理放在空间和时间上下文中 迁移细胞。对于PI3K途径，我们将分析控制PIP3定位的蛋白质 磷酸酶PTEN到细胞背面的质膜。 PTEN的后部定位启用 PIP3信号在前缘处的激活及其在后端的抑制作用。我们还将确定 控制PTEN定位在肿瘤发生和转移中的确定机制的功能 在表达具有改变定位的工程PTEN分子的小鼠异种移植模型中。这些 研究将阐明细胞内信号的极化为基本逻辑 在趋化性迁移过程中建立在细胞中的细胞和生理重要性 体内信号传导。