Regulation of Chemotactic Signaling

趋化信号的调节

基本信息

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
项目类别：
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

项目摘要

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，异常的趋化性会导致慢性炎症性疾病和肿瘤转移。我的实验室长期以来一直对控制细胞趋化行为的信号机制感兴趣。在在化学梯度下，细胞极化细胞内信号并向化学引诱剂迁移。配体与趋化受体结合选择性激活位于趋化因子前端的两种激酶：TORC2 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受体的下游立即发挥作用。极化激活这两种激酶对于创造在化学物质中迁移的细胞的前沿至关重要坡度。尽管与基础科学和医学科学至关重要，但对空间和空间的理解 TORC2 和 PI3K 的时间调节仍然不完整。首要的挑战是破译 TORC2 和 PI3K 信号通路如何在细胞的前部和后部受到调节化学梯度。为了探索调节 TORC2 通路的机制，我们将利用我们最近的研究成果开发了生化系统。在这些系统中，趋化剂调节的激活和抑制 TORC2 是用纯化的 TORC2 和两个小 GTP 酶 Rho 和 Ras 忠实重建的。这些新的测定将确定产生不同趋化性的生化和生物物理机制迁移细胞前缘和后缘的信号传导。确定的机械原理将使用表达的敲除细胞系在我们的细胞重建系统中进行测试和翻译 WT 和突变体 TORC2 及其调控组件。使用 FRET 显微镜进行活细胞成像和单分子显微镜，我们将信号原理置于空间和时间背景中迁移细胞。对于 PI3K 通路，我们将分析控制 PIP3 定位的蛋白质磷酸酶 PTEN 作用于细胞背面的质膜。 PTEN 的后部定位使得 PIP3 信号在前缘激活，在后缘抑制。我们还将确定已确定的控制 PTEN 定位在肿瘤发生和转移中的机制的功能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迁移过程中在细胞中建立及其生理重要性体内信号传导。