Real-time imaging of notch activation with a luciferase complementation-based reporter.
Real-time imaging of notch activation with a luciferase complementation-based reporter.
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DOI：
10.1126/scisignal.2001656
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发表时间：
2011-07-12
影响因子：
7.3
通讯作者：
Kopan R
中科院分区：
文献类型：
--
作者：
Ilagan MX;Lim S;Fulbright M;Piwnica-Worms D;Kopan R
Notch信号调节发育和成体组织更新过程中的许多细胞过程。在配体结合后，Notch受体经历胞外域脱落，随后是γ-分泌酶介导的Notch胞内结构域（NICD）的释放，其易位至细胞核并与DNA结合蛋白CSL（CBF 1/RBPjκ/Su（H）/Lag 1）结合以激活基因表达。哺乳动物细胞含有四种Notch受体，可以具有冗余和特异性活性。为了监测活细胞中特异性Notch旁系同源物的激活和真实的时间，我们开发了用于NICD/CSL关联的荧光素酶互补成像（LCI）报告基因，并将其验证为特异性、稳健性和灵敏性的测定系统，其能够进行结构-功能和药效学分析。在活细胞中进行Notch信号传导的各种机制方面的详细动力学分析，包括核转位、γ-分泌酶和ADAM抑制以及激动剂和配体依赖性活化。Notch-LCI代表了一种强有力的方法，用于表征靶向Notch信号传导的调节剂，并用于研究正常和疾病背景下的通路动力学。
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, γ-secretase and ADAM inhibition, as well as agonist- and ligand-dependent activation were conducted in live cells. Notch-LCI represents a powerful approach for characterizing modulators that target Notch signaling and for studying pathway dynamics in normal and disease contexts.
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影响因子：
3.7
作者：
Gordon WR;Vardar-Ulu D;L'Heureux S;Ashworth T;Malecki MJ;Sanchez-Irizarry C;McArthur DG;Histen G;Mitchell JL;Aster JC;Blacklow SC
通讯作者：
Blacklow SC
影响因子：
4.8
作者：
Chavez-Gutierrez, Lucia;Tolia, Alexandra;de Strooper, Bart
通讯作者：
de Strooper, Bart
影响因子：
5.3
作者：
Jeffries, S;Robbins, DJ;Capobianco, AJ
通讯作者：
Capobianco, AJ
影响因子：
5.3
作者：
Bozkulak, Esra Cagavi;Weinmaster, Gerry
通讯作者：
Weinmaster, Gerry
影响因子：
2.1
作者：
Kawahashi, Koji;Hayashi, Shigeo
通讯作者：
Hayashi, Shigeo