Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells
Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells
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DOI：
10.1039/c9pp00249a
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发表时间：
2019-11-01
影响因子：
3.1
通讯作者：
Ozawa，Takeaki
中科院分区：
文献类型：
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作者：
Noda，Natsumi;Ishimoto，Tetsuya;Ozawa，Takeaki
环磷酸腺苷(CAMP)反应元件结合蛋白(CREB)与记忆形成有关，并通过调节肿瘤发生过程中下游基因的表达来控制细胞的存活和增殖。作为一种转录因子，CREB与cAMP反应元件结合。CREB的磷酸化触发CREB下游基因的转录激活，CREB的激酶诱导结构域(KID)与CREB结合蛋白的KID相互作用结构域(KIX)相互作用。然而，由于缺乏单细胞分析技术，对CREB磷酸化的时空调控知之甚少。为了分析单个活细胞中CREB的激活，我们利用荧光素酶片段互补技术开发了基因编码的生物发光传感器：这些传感器是基于KID-KIX相互作用设计的单分子形式。该传感器被命名为CREX(一种基于KID(CREB)-KIX相互作用的CREB激活传感器)，通过CREB的磷酸化而增加其发光强度。此外，CRIX的发光强度足以在活细胞生物发光成像中检测CREB的激活，因为它具有较高的灵敏度。CREX传感器有望通过应用单细胞分析来阐明CREB磷酸化的时空调控。
Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferationviaregulation 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.