Westwick John K, Michnick Stephen W
Canada Research Chair in Integrative Genomics, Département de Biochimie, Université de Montreal, Canada.
Methods Enzymol. 2006;407:388-401. doi: 10.1016/S0076-6879(05)07032-1.
Small GTPases of the Ras and Rho families are among the most studied signaling proteins and represent promising therapeutic targets for human neoplastic disease. Despite the high level of interest in these proteins, direct analysis of most aspects of Ras protein biology in living cells has not been possible, because much of the details of Ras signaling cannot be studied in vitro but requires simple cell-based assays. Here we describe a strategy for directly analyzing Ras signaling pathways in living cells using protein-fragment complementation assays (PCA) based on fragments of intensely fluorescent proteins. The assays allow for spatial and temporal analysis of protein complexes including those that form upstream and downstream from Ras proteins, as well as complexes of Ras proteins with regulator and effector proteins. We describe high-throughput quantitative microscopic methods to follow temporal changes in complex subcellular location and quantity (high-content assays). Spatial and temporal changes in response to perturbations (chemical, siRNA, hormones) allow for delineation of Ras signaling networks and a general and high-throughput approach to identify drugs that act directly or indirectly on Ras pathways.
Ras和Rho家族的小GTP酶是研究最多的信号蛋白之一，也是人类肿瘤疾病有前景的治疗靶点。尽管对这些蛋白高度关注，但由于Ras蛋白生物学的大多数方面无法在体外研究，而需要基于简单细胞的检测方法，因此在活细胞中直接分析Ras蛋白生物学的大多数方面是不可能的。在这里，我们描述了一种基于强荧光蛋白片段，使用蛋白质片段互补分析（PCA）在活细胞中直接分析Ras信号通路的策略。这些分析允许对蛋白质复合物进行空间和时间分析，包括那些在Ras蛋白上游和下游形成的复合物，以及Ras蛋白与调节蛋白和效应蛋白的复合物。我们描述了高通量定量显微镜方法，以跟踪复杂亚细胞定位和数量的时间变化（高内涵分析）。对扰动（化学物质、siRNA、激素）的空间和时间变化允许描绘Ras信号网络，并提供一种通用的高通量方法来识别直接或间接作用于Ras通路的药物。
