Regulation of Chemotactic Signaling

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。我的实验室 长期以来一直对控制细胞趋化性行为的信号机制感兴趣。某化工 在梯度下，细胞抑制细胞内信号传导并向化学引诱物迁移。配体结合 化学引诱物受体选择性地激活细胞前部的两种激酶TORC 2和PI3K。TORC2 和PI3K是诱导细胞骨架重塑以延长伪足和功能的主要激酶 就在趋化性受体的下游。这两种激酶的极化激活是必不可少的 用于产生以化学梯度迁移的细胞的前缘。尽管这与 基础和医学科学，了解TORC 2和PI3K的时空调节 仍然不完整。一个首要的挑战是破译TORC 2和PI3K信号通路是如何 在细胞的前部和后部以化学梯度进行调节。 为了探索调节TORC 2通路的机制，我们将利用我们最近开发的 生化系统在这些系统中，趋化因子调节的TORC 2的激活和抑制被抑制。 用纯化的TORC 2和两种小GTP酶Rho和Ras忠实地重构。这些新的检测方法将 确定生物化学和生物物理机制，创造不同的趋化性信号的领导 和迁移细胞的后缘。所确定的机械原理将被测试和翻译 在我们的细胞重建系统中，使用表达WT和突变型TORC 2的敲除细胞系， 监管成分。使用活细胞成像与FRET显微镜和单分子显微镜，我们将 将信号原理置于迁移细胞的空间和时间背景中。对于PI3K通路，我们将 分析控制PIP3磷酸酶PTEN在细胞膜上定位的蛋白质， 细胞的背面。PTEN的后部定位使得PIP3信号传导在前沿激活， 抑制在后缘。我们还将确定已识别的控制机制的功能 PTEN基因在小鼠移植瘤发生和转移中的定位 改变了定位的分子。这些研究将阐明基本逻辑， 在趋化性迁移期间在细胞中建立细胞内信号传导的极化， 这种信号在体内的生理重要性。

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.