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Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells†
Photochemical & Photobiological Sciences ( IF 3.1 ) Pub Date : 2019-09-21 , DOI: 10.1039/c9pp00249a
Natsumi Noda 1, 2, 3, 4, 5 , Tetsuya Ishimoto 5, 6, 7, 8, 9 , Hisashi Mori 5, 6, 7, 8, 9 , Takeaki Ozawa 1, 2, 3, 4, 5
Affiliation  

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID–KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)–KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.

中文翻译:

增强型生物发光传感器,用于纵向检测活细胞中的CREB激活

环状单磷酸腺苷(cAMP)反应元件结合蛋白(CREB)与记忆形成有关,并通过以下途径控制细胞存活和增殖肿瘤发生中下游基因表达的调控。作为转录因子,CREB与cAMP反应元件结合。在CREB的激酶诱导域(KID)与CREB结合蛋白的KID相互作用域(KIX)相互作用之后,CREB的磷酸化触发CREB下游基因的转录激活。然而,由于缺乏单细胞分析技术,对CREB磷酸化的时空调节知之甚少。为了分析单个活细胞中的CREB活化,我们开发了使用荧光素酶片段互补技术的基因编码生物发光传感器:该传感器是基于KID-KIX与单分子形式的相互作用而设计的。传感器的发光强度,称为CREX(基于KID(CREB)–KIX相互作用的CREB激活传感器),通过CREB的磷酸化增加。此外,由于具有更高的灵敏度,CREX的发光强度足以检测活细胞生物发光成像中的CREB激活,从而进行单细胞分析。通过应用单细胞分析,预计CREX传感器将有助于阐明CREB磷酸化的时空调控。
更新日期:2019-11-06
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