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.

总结 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性疾病和肿瘤转移。我 长期以来，实验室一直对控制细胞趋化性行为的信号机制感兴趣。在 在化学梯度下，细胞抑制细胞内信号传导并向化学引诱物迁移。配体 与化学引诱物受体结合，选择性地激活两种激酶，TORC 2和PI3K， 细胞TORC2和PI3K是诱导细胞骨架重塑以延长细胞周期的主要激酶。 伪足并在趋化性受体的下游立即起作用。极化激活 这两种激酶对于创造细胞的前沿是必不可少的， 梯度离心尽管与基础科学和医学科学至关重要，但对空间和 TORC2和PI3K的时间调节仍然不完全。最大的挑战是破解 TORC2和PI3K信号通路是如何在细胞的前部与后部进行调节的， 化学梯度。 为了探索调节TORC 2通路的机制，我们将利用我们最近的研究成果， 发展了生化系统。在这些系统中，趋化因子调节的激活和抑制 TORC2用纯化的TORC2和两种小GTP酶Rho和Ras忠实地重构。这些 新的分析将确定产生不同趋化性的生物化学和生物物理机制， 在迁移细胞的前沿和后沿发出信号。机械原理决定了 将在我们的细胞重建系统中进行测试和翻译， WT和突变型T0RC2及其调控组分。使用FRET显微镜的活细胞成像 和单分子显微镜，我们将把信号原理在空间和时间的背景下， 迁移细胞对于PI 3 K途径，我们将分析控制PIP 3定位的蛋白质 磷酸酶PTEN与细胞背面的质膜结合。PTEN的后部定位使得 PIP3信号在前沿激活，在后沿抑制。我们还将确定 已鉴定的控制PTEN定位的机制在肿瘤发生和转移中的作用 在表达具有改变的定位的工程化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极化的基本逻辑， 在趋化性迁移过程中在细胞中建立， 体内信号传导