Regulation of Chemotactic Signaling

摘要 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 长期以来一直对控制细胞趋化行为的信号机制感兴趣。在一种化学品中 梯度，细胞极化细胞内的信号，并向化学诱导剂迁移。配体结合到 趋化受体选择性地激活细胞前部的两个激酶--TORC2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 就在趋化受体的下游。这两种酶的极化激活是必不可少的。 用于创造在化学梯度中迁移的细胞的前沿。尽管与 基础和医学，了解TORC2和PI3K的时空调节 仍然不完整。最大的挑战是破译TORC2和PI3K信号通路 在化学梯度中被调节在细胞的前部和后部。 为了探索调控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，化学吸引物调节的TORC2的激活和抑制是 与纯化的TORC2和两个小的GTP酶Rho和RAS忠实地重组。这些新的化验方法将 确定在前导产生不同趋化信号的生化和生物物理机制 以及迁移细胞的后缘。确定的机械原理将被测试和翻译 在我们使用表达WT和突变体TORC2及其基因的敲除细胞系的细胞重建系统中 监管组成部分。使用活细胞成像与FRET显微镜和单分子显微镜，我们将 将信号传递原理放在迁移细胞的空间和时间上下文中。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 牢房的背面。PTEN的后部定位使PIP3信号在前沿激活，并在其 在后缘的抑制。我们还将确定已确定的控制机制的功能 PTEN基因在小鼠异种移植瘤发生和转移中的定位 局部化改变的分子。这些研究将阐明以下基本逻辑： 在趋化迁移过程中，细胞内信号的极化是建立在细胞内的 这一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.