Real-time imaging of ß-catenin dynamics in cells and living mice
Real-time imaging of ß-catenin dynamics in cells and living mice
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DOI：
10.1073/pnas.0704465104
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发表时间：
2007-10-30
影响因子：
11.1
通讯作者：
Piwnica-Worms, David
中科院分区：
文献类型：
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作者：
Naik, Snehal;Piwnica-Worms, David
β-连环蛋白（β-cat）是经典Wnt通路的关键信号传导组分，也是调节细胞粘附、增殖和分化的各种通路的越来越多研究的贡献者。对于其最著名的功能，β-cat的翻译后稳定是Wnt的许多下游靶点的T细胞因子依赖性转录所必需的，并且该过程在多种癌症中异常活跃。为了能够直接监测β-cat在细胞和活体动物中的翻译后稳定性，我们构建并表征了生物发光融合报告基因β-cat萤火虫荧光素酶（β-cat-FLuc）和β-cat甲虫绿色荧光素酶（β-cat-CEIG）。这些报告者提供了实时、非侵入性的β-cat在细胞中稳定性调节剂的读数，此外，还能够监测完整动物体内总β-cat水平的变化。此外，使用光谱解混，将绿色β-cat-CBG从红色TCF依赖性FLuc报告基因（TOPFLASH）解卷积，使得能够同时分析β-cat加工和下游转录激活。通过使用该系统，发现天然产物表没食子儿茶素3-没食子酸酯阻断Wnt信号传导，而不依赖于β-cat加工。这些β-cat报告代表了一种强大的新策略，用于在细胞内和体内鉴定由β-cat稳定化介导的信号传导途径的动态调节剂和基于机制的治疗剂。
beta-Catenin (beta-cat) is a key signaling component of the canonical Wnt pathway as well as an increasingly studied contributor to various pathways that regulate cell adhesion, proliferation, and differentiation. For its best known function, posttranslational stabilization of beta-cat is required for T cell factor-dependent transcription of numerous downstream targets of Wnt, and this process is aberrantly active in a wide array of cancers. To enable direct monitoring of posttranslational stabilization of beta-cat in cells and living animals, we constructed and characterized the bioluminescent fusion reporters beta-cat firefly lucif erase (beta-cat-FLuc) and beta-cat click beetle green luciferase (beta-cat-CEIG). These reporters provided real-time, noninvasive readout of modulators of beta-cat stability in cellulo and, furthermore, enabled monitoring of changes in total beta-cat levels in vivo in intact animals. in addition, using spectral unmixing, green beta-cat-CBG was deconvoluted from a red TCF-dependent FLuc reporter (TOPFLASH), enabling analysis of beta-cat processing and downstream transcriptional activation simultaneously. By using this system, the natural product epigallocatechin 3-gallate was found to block Wnt signaling, independent of beta-cat processing. These beta-cat reporters represent a powerful new strategy for identifying in cellulo and in vivo dynamic regulators and mechanism-based therapeutics of signaling pathways mediated by beta-cat stabilization.