Quantifying E2F1 protein dynamics in single cells.
Quantifying E2F1 protein dynamics in single cells.
复制标题
量化单细胞中的 E2F1 蛋白动态。
DOI：
10.1007/s40484-019-0193-6
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
You,Lingchong
中科院分区：
文献类型：
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作者：
Mathey-Prevot,Bernard;Parker,Bao-Tran;Im,Carolyn;Hong,Cierra;Dong,Peng;Yao,Guang;You,Lingchong
研究背景E2 F1蛋白是Rb/E2 F信号通路的主要效应子，在细胞增殖、凋亡和分化过程中起着重要的调控作用。它的表达是高度动态的，并通过转录，翻译和翻译后控制的组合进行紧密调节。然而，其表达和活性可促进不同细胞结果的机制仍有待充分阐明。为了更好地记录E2 F1在活细胞中的表达，我们设计了一系列荧光E2 F1蛋白报告分子，定量捕获E2 F1蛋白dynamicsMethodsReporter构建体，在小鼠或人E2 F1近端启动子的控制下，设计用于表达不能结合DNA的E2 F1-Venus融合蛋白。此外，构建体包括或不包括E2 F1基因的3′非翻译区（3′UTR）。这些构建体被引入成纤维细胞和上皮细胞，融合报告蛋白的表达进行了验证和定量在单细胞使用live imaging.ResultsIn所有情况下，报告蛋白的表达有效地概括了E2 F1在各种条件下的行为，包括细胞周期进展和遗传毒性应激。在G 0中没有或几乎没有检测到报告基因的荧光信号，但随着周期的进行，报告基因蛋白的表达在细胞核中稳定增加，在细胞分裂前几小时达到峰值，但在有丝分裂开始前2-3小时下降至基线。构建体中E2 F1 3′UTR的缺失导致融合蛋白的稳态水平显著较高，尽管其正常调节，但在细胞周期或遗传毒性应激期间表现出稍微不那么复杂的动态特征。最后，Rb的存在或不存在未能影响的整体检测和水平的报告proteins.ConclusionsOur验证的E2 F1蛋白的报告很好地补充Rb/E2 F途径的其他报告，并提供了一个独特的工具，遵循复杂的动态E2 F1表达在真实的时间在单细胞。
BackgroundE2F1 protein, a major effector of the Rb/E2F pathway plays a central role in regulating cell‐fate decisions involved in proliferation, apoptosis, and differentiation. Its expression is highly dynamic and tightly modulated through a combination of transcriptional, translational and posttranslational controls. However, the mechanisms by which its expression and activity can promote different cellular outcomes remain to be fully elucidated. To better document E2F1 expression in live cells, we have engineered a series of fluorescent E2F1 protein reporters that quantitatively capture E2F1 protein dynamics.MethodsReporter constructs, under the control of the mouse or human E2F1 proximal promoter, were designed to express an E2F1‐Venus fusion protein incapable of binding DNA. In addition, constructs either included or excluded the 3′ untranslated region (3′UTR) of theE2F1gene. These constructs were introduced into fibroblasts and epithelial cells, and expression of the fusion reporter protein was validated and quantified in single cells using live imaging.ResultsIn all cases, expression of the reporter protein effectively recapitulated the behavior of E2F1 under various conditions, including cell cycle progression and genotoxic stress. No or little fluorescent signal of the reporter was detected in G0, but as the cycle progressed, expression of the reporter protein steadily increased in the nucleus, peaking a few hours before cell division, but declining to baseline 2–3 h prior to the onset of mitosis. The absence of the E2F1 3′UTR in the constructs led to considerably higher steady‐state levels of the fusion protein, which although normally regulated, exhibited a slightly less complex dynamic profile during the cell cycle or genotoxic stress. Lastly, the presence or absence of Rb failed to impact the overall detection and levels of the reporter proteins.ConclusionsOur validated E2F1 protein reporters complement nicely other reporters of the Rb/E2F pathway and provide a unique tool to follow the complex dynamics of E2F1 expression in real time in single cells.