Visualizing cellular heterogeneity by quantifying the dynamics of MAPK activity in live mammalian cells with synthetic fluorescent biosensors.
Visualizing cellular heterogeneity by quantifying the dynamics of MAPK activity in live mammalian cells with synthetic fluorescent biosensors.
复制标题
DOI：
10.1016/j.heliyon.2020.e05574
复制
发表时间：
2020-12
期刊：
影响因子：
4
通讯作者：
Pelet S
中科院分区：
文献类型：
--
作者：
Ma M;Bordignon P;Dotto GP;Pelet S
丝裂原活化蛋白激酶（MAPKs）通过将细胞外信息转导为定义的生物反应来控制广泛的细胞功能。为了了解这些途径是如何被调节的，动态的单细胞测量是非常必要的。荧光显微镜非常适合执行这些测量。然而，需要更多动态和敏感的生物传感器来量化活哺乳动物细胞中的信号活动。我们设计了一种合成的人类mapk （ERK， JNK和p38）荧光底物，当被激酶磷酸化时，这些mapk会从细胞核迁移到细胞质中。我们证明，与其他定位生物传感器相比，该报告显示了改进的响应。该试验可以监测等基因细胞群体中MAPK反应的异质性，揭示生理EGFR刺激时ERK活性的脉冲。我们展示了这种方法对多种癌细胞系和原代细胞分析的适用性，以及它在体内发展肿瘤的应用。使用这种ERK生物传感器，可以获得高时间分辨率的动态单细胞测量。这些MAPK报告基因可广泛应用于健康和疾病状态、细胞培养试验或体内MAPK信号传导的分子机制分析。细胞生物学;系统生物学;生物化学;癌症研究;MAPK信号;单个细胞;荧光生物传感器;Live-cell成像
Mitogen-Activated Protein Kinases (MAPKs) control a wide array of cellular functions by transducing extracellular information into defined biological responses. In order to understand how these pathways are regulated, dynamic single cell measurements are highly needed. Fluorescence microscopy is well suited to perform these measurements. However, more dynamic and sensitive biosensors that allow the quantification of signaling activity in living mammalian cells are required. We have engineered a synthetic fluorescent substrate for human MAPKs (ERK, JNK and p38) that relocates from the nucleus to the cytoplasm when phosphorylated by the kinases. We demonstrate that this reporter displays an improved response compared to other relocation biosensors. This assay allows to monitor the heterogeneity in the MAPK response in a population of isogenic cells, revealing pulses of ERK activity upon a physiological EGFR stimulation. We show applicability of this approach to the analysis of multiple cancer cell lines and primary cells as well as its application in vivo to developing tumors. Using this ERK biosensor, dynamic single cell measurements with high temporal resolution can be obtained. These MAPK reporters can be widely applied to the analysis of molecular mechanisms of MAPK signaling in healthy and diseased state, in cell culture assays or in vivo. Cell biology; Systems biology; Biochemistry; Cancer research; MAPK signaling; Single cells; Fluorescent biosensor; Live-cell imaging
登录
查看更多内容
影响因子：
16
作者：
Cohen-Saidon, Cellina;Cohen, Ariel A.;Alon, Uri
通讯作者：
Alon, Uri
DOI：
10.1073/pnas.0909671107
发表时间：
2010-03-23
影响因子：
11.1
作者：
Fosbrink, Matthew;Aye-Han, Nwe-Nwe;Zhang, Jin
通讯作者：
Zhang, Jin
影响因子：
64.5
作者：
Altschuler SJ;Wu LF
通讯作者：
Wu LF
影响因子：
16
作者：
Chang, CI;Xu, BE;Goldsmith, EJ
通讯作者：
Goldsmith, EJ
影响因子：
4.8
作者：
Hodel, MR;Corbett, AH;Hodel, AE
通讯作者：
Hodel, AE