Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression
Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression
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DOI：
10.1247/csf.16007
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发表时间：
2016-01-01
影响因子：
1.5
通讯作者：
Aoki, Kazuhiro
中科院分区：
文献类型：
--
作者：
Maryu, Gembu;Matsuda, Michiyuki;Aoki, Kazuhiro
Ras-ERK通路控制细胞增殖和分化，而PI 3 K-Akt通路在细胞周期进展和细胞存活过程中起作用。这两种途径都被许多刺激物激活，如表皮生长因子（EGF），并通过串扰相互协调调节。然而，目前尚不清楚细胞如何调节Ras-ERK和PI 3 K-Akt通路之间的动力学和相互作用以调节细胞命运决定，主要是因为缺乏良好的工具来同时在活细胞中可视化ERK和Akt活性。在这里，我们开发了一个多重荧光系统，用于在单细胞水平上成像ERK和Akt信号和细胞周期状态。基于激酶易位报告基因（KTR）的原理，我们构建了Akt-FoxO 3a-KTR，它在细胞核和细胞质之间穿梭，受Akt磷酸化的调节。为了同时测量ERK、Akt和细胞周期状态，我们产生了表达ERK-KTR、Akt-FoxO 3a-KTR、细胞周期报告基因和核报告基因的多顺反子载体，并对这四个图像应用线性解混以去除荧光蛋白之间的光谱重叠。定量分析ERK-KTR和Akt-FoxO 3a-KTR的特异性和敏感性。我们研究了ERK和Akt活性在基础或EGF刺激条件下的细胞异质性关系，发现ERK和Akt以高度协同和细胞周期依赖的方式调节。我们的研究提供了一个有用的工具，定量ERK和Akt活性和细胞周期之间的动态在活细胞，并解决内在的耐药分子靶向药物的机制。
The Ras-ERK pathway controls cell proliferation and differentiation, whereas the PI3K-Akt pathway plays a role in the process of cell-cycle progression and cell survival. Both pathways are activated by many stimuli such as epidermal growth factor (EGF), and coordinately regulate each other through cross-talk. However, it remains unclear how cells accommodate the dynamics and interplay between the Ras-ERK and PI3K-Akt pathways to regulate cell-fate decisions, mainly because of the lack of good tools to visualize ERK and Akt activities simultaneously in live cells. Here, we developed a multiplexed fluorescence system for imaging ERK and Akt signaling and the cell-cycle status at the single cell level. Based on the principle of the kinase translocation reporter (KTR), we created Akt-FoxO3a-KTR, which shuttled between nucleus and cytoplasm in a manner regulated by Akt phosphorylation. To simultaneously measure ERK, Akt and the cell-cycle status, we generated a polycistronic vector expressing ERK-KTR, Akt-FoxO3a-KTR, a cell-cycle reporter and a nuclear reporter, and applied linear unmixing to these four images to remove spectral overlap among fluorescent proteins. The specificity and sensitivity of ERK-KTR and Akt-FoxO3a-KTR were characterized quantitatively. We examined the cellular heterogeneity of relationship between ERK and Akt activities under a basal or EGF-stimulated condition, and found that ERK and Akt were regulated in a highly cooperative and cell-cycledependent manner. Our study provides a useful tool for quantifying the dynamics among ERK and Akt activities and the cell cycle in a live cell, and for addressing the mechanisms underlying intrinsic resistance to molecularly targeted drugs.