Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Sci Signal. 2011 Jul 12;4(181):rs7. doi: 10.1126/scisignal.2001656.
Notch signaling regulates many cellular processes during development and adult tissue renewal. Upon ligand binding, Notch receptors undergo ectodomain shedding followed by γ-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA binding protein CSL [CBF1/RBPjκ/Su(H)/Lag1] to activate gene expression. Mammalian cells contain four Notch receptors that can have both redundant and specific activities. To monitor activation of specific Notch paralogs in live cells and in real time, we developed luciferase complementation imaging (LCI) reporters for NICD-CSL association and validated them as a specific, robust, and sensitive assay system that enables structure-function and pharmacodynamic analyses. Detailed kinetic analyses of various mechanistic aspects of Notch signaling, including nuclear translocation and inhibition of the activities of γ-secretase and ADAM metalloproteases, as well as agonist- and ligand-dependent activation, were conducted in live cells. These experiments showed that Notch-LCI is an effective approach for characterizing modulators that target Notch signaling and for studying pathway dynamics in normal and disease contexts.
Notch 信号通路在发育和成人组织更新过程中调节许多细胞过程。配体结合后，Notch 受体经历外显子脱落，随后由 γ-分泌酶介导释放 Notch 细胞内结构域（NICD），NICD 易位到细胞核并与 DNA 结合蛋白 CSL [CBF1/RBPjκ/Su(H)/Lag1]结合以激活基因表达。哺乳动物细胞包含四个 Notch 受体，它们具有冗余和特定的活性。为了在活细胞中实时监测特定 Notch 同源物的激活，我们开发了用于 NICD-CSL 结合的荧光素酶互补成像（LCI）报告基因，并将其验证为一种特异性、稳健且敏感的测定系统，能够进行结构-功能和药效动力学分析。在活细胞中对 Notch 信号转导的各种机制方面进行了详细的动力学分析，包括核易位以及 γ-分泌酶和 ADAM 金属蛋白酶活性的抑制，以及激动剂和配体依赖性激活。这些实验表明，Notch-LCI 是一种有效的方法，可用于表征针对 Notch 信号转导的调节剂，并用于研究正常和疾病情况下的途径动态。
