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 长期以来，实验室一直对控制细胞趋化行为的信号机制感兴趣。在……里面 作为一种化学梯度，细胞极化细胞内的信号并向趋化物质迁移。配基 与趋化受体的结合选择性地激活了TORC2和PI3K这两个位于 细胞。TORC2和PI3K是诱导细胞骨架重塑延长的主要激酶 伪足和功能就在趋化受体的下游。极化激活 这两种激酶对于形成细胞在化学物质中迁移的前沿是必不可少的。 渐变。尽管与基础和医学科学密切相关，但对空间和 TORC2和PI3K的时间调控仍然不完整。最大的挑战是破译 TORC2和PI3K信号通路是如何在细胞的前部和后部进行调节的 一种化学梯度。 为了探索调控TORC2途径的机制，我们将利用我们最近的 开发了生化系统。在这些系统中，化学诱导剂调节的激活和抑制 TORC2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产生不同趋化性的生化和生物物理机制 在迁移细胞的前沿和后缘发出信号。被决定的机械论原理 将在我们的细胞重建系统中使用敲除细胞系进行测试和翻译，表达 WT和突变体TORC2及其调控元件。使用FRET显微镜进行活细胞成像 和单分子显微镜，我们将把信号原理放在空间和时间的背景下 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连接到细胞背面的质膜上。PTEN的后部本地化使 PIP3信号在前沿被激活，在下降沿被抑制。我们还将确定 已发现的控制PTEN定位的机制在肿瘤发生和转移中的作用 在小鼠异种移植模型中，表达定位改变的工程化PTEN分子。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的建立及其生理意义 体内的信号。

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.