Regot Sergi, Hughey Jacob J, Bajar Bryce T, Carrasco Silvia, Covert Markus W
Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
Cell. 2014 Jun 19;157(7):1724-34. doi: 10.1016/j.cell.2014.04.039.
Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.
越来越多的证据表明，群体动态在性质上不同于单细胞行为。用于探测动态单细胞行为的报告分子是很有必要的，但相对较少。在这里，我们描述了一种易于实施且可推广的技术，用于生成单个细胞激酶活性的报告分子。我们的技术将磷酸化转化为可通过落射荧光显微镜测量的核质穿梭事件。我们的报告分子可重现多种类型激酶的激酶活性，并允许通过数学模型计算活性激酶浓度。利用这项技术，我们进行了一些以前在技术上不可行的实验观察，包括c-Jun氨基末端激酶（JNK）和核因子κB（NF-κB）激活的刺激依赖性模式。我们还同时测量了JNK、p38和ERK的活性，发现p38调节ERK波动的峰值数量，但不调节其强度。我们的方法为分析单个细胞中广泛的激酶介导过程开辟了可能性。
