Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor
Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor
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DOI：
10.1073/pnas.0909671107
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发表时间：
2010-03-23
影响因子：
11.1
通讯作者：
Zhang, Jin
中科院分区：
文献类型：
--
作者：
Fosbrink, Matthew;Aye-Han, Nwe-Nwe;Zhang, Jin
JNK介导的信号通路将不同类型的信号，如应激刺激和细胞因子，转化为介导细胞凋亡、增殖、分化和炎症的功能反应。为了更好地表征这一途径在细胞环境中的动态信息流和信号处理，设计了一种基于基因编码的基于荧光蛋白的生物传感器来检测内源性JNK活性。这种名为JNKAR1(JNK Activity Report)的生物传感器可以特异性地检测应激(核毒和渗透)和细胞因子(TNF-α)诱导的活细胞JNK活性，由于两种荧光蛋白之间的FRET依赖于磷酸化增加，黄蓝发射比增加了15%到30%。苯甲酸诱导核毒应激后，JNK活性不仅在细胞质中检测到，而且在细胞核、线粒体和质膜中也以相似的动力学方式检测到，这表明信号在核、线粒体和质膜中的传播相对较快。此外，定量的单细胞分析表明，山奈霉素诱导的JNK活性具有超高的敏感性、持续性和双峰性，这些特征与双稳态系统的行为相一致。因此，JNKAR1的发展为评价JNK级联在单个活细胞中的信号特性和行为奠定了基础。
The signaling pathway mediated by JNK transduces different types of signals, such as stress stimuli and cytokines, into functional responses that mediate apoptosis, as well as proliferation, differentiation, and inflammation. To better characterize the dynamic information flow and signal processing of this pathway in the cellular context, a genetically encoded, fluorescent protein-based biosensor was engineered to detect endogenous JNK activity. This biosensor, named JNKAR1 (for JNK activity reporter), specifically detects stress-(ribotoxic and osmotic) and cytokine-(TNF-alpha) induced JNK activity in living cells with a 15 to 30% increase in the yellow-to-cyan emission ratio because of a phosphorylation-dependent increase in FRET between two fluorescent proteins. JNK activity was detected not only in the cytoplasm, but also in the nucleus, mitochondria, and plasma membrane with similar kinetics after induction of ribotoxic stress by anisomycin, suggesting relatively rapid signal propagation to the nuclear, mitochondrial, and plasma membrane compartments. Furthermore, quantitative single-cell analysis revealed that anisomycin-induced JNK activity exhibited ultrasensitivity, sustainability, and bimodality, features that are consistent with behaviors of bistable systems. The development of JNKAR1, therefore, laid a foundation for evaluating the signaling properties and behaviors of the JNK cascade in single living cells.