High-throughput and single-cell imaging of NF-kappaB oscillations using monoclonal cell lines.
High-throughput and single-cell imaging of NF-kappaB oscillations using monoclonal cell lines.
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DOI：
10.1186/1471-2121-11-21
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发表时间：
2010-03-16
期刊：
影响因子：
--
通讯作者：
Meyer TF
中科院分区：
文献类型：
--
作者：
Bartfeld S;Hess S;Bauer B;Machuy N;Ogilvie LA;Schuchhardt J;Meyer TF
核转录因子-κB（NF-κB）家族在包括免疫应答和细胞生长在内的广泛的细胞过程中发挥作用。此外，NF-κB系统的失调与许多疾病状态相关，包括癌症。因此，深入了解NF-κB活化的调节具有重要的医学意义，为发现新的药物靶点带来了希望。NF-κ B诱导的基因转录受单个NF-κB亚基的差异动力学调节，但目前只有少数方法用于研究动力学。特别是，虽然已经观察到NF-κB活化的振荡响应于细胞因子肿瘤坏死因子α（TNFα），但对振荡响应于细菌感染的发生知之甚少。为了定量评估NF-κB动力学，我们产生了稳定表达与GFP融合的NF-κB亚基p65的人和鼠单克隆细胞系。此外，建立了一种基于自动显微镜结合图像分析的高通量检测方法来定量p65核转位。使用这种方法，我们证明了刺激和细胞系特异性的时间控制p65易位，揭示了，第一次，振荡p65易位响应细菌感染。振荡检测在单细胞水平上使用实时显微镜，以及在人口水平上使用高通量图像分析。此外，细菌感染期间NF-κB动力学的数学建模预测了异步激活中群体水平上振荡的掩蔽，这一点得到了实验证实。综上所述，这种简单且具有成本效益的测定构成了推断单个细胞和细胞群体中NF-κB动力学的综合方法。使用单一系统，可以鉴定和分析调节NF-κB的新因子，为从疾病的治疗发现和理解到宿主-病原体相互作用的广泛应用提供了新的可能性。
The nuclear factor-κB (NF-κB) family of transcription factors plays a role in a wide range of cellular processes including the immune response and cellular growth. In addition, deregulation of the NF-κB system has been associated with a number of disease states, including cancer. Therefore, insight into the regulation of NF-κB activation has crucial medical relevance, holding promise for novel drug target discovery. Transcription of NF-κB-induced genes is regulated by differential dynamics of single NF-κB subunits, but only a few methods are currently being applied to study dynamics. In particular, while oscillations of NF-κB activation have been observed in response to the cytokine tumor necrosis factor α (TNFα), little is known about the occurrence of oscillations in response to bacterial infections. To quantitatively assess NF-κB dynamics we generated human and murine monoclonal cell lines that stably express the NF-κB subunit p65 fused to GFP. Furthermore, a high-throughput assay based on automated microscopy coupled to image analysis to quantify p65-nuclear translocation was established. Using this assay, we demonstrate a stimulus- and cell line-specific temporal control of p65 translocation, revealing, for the first time, oscillations of p65 translocation in response to bacterial infection. Oscillations were detected at the single-cell level using real-time microscopy as well as at the population level using high-throughput image analysis. In addition, mathematical modeling of NF-κB dynamics during bacterial infections predicted masking of oscillations on the population level in asynchronous activations, which was experimentally confirmed. Taken together, this simple and cost effective assay constitutes an integrated approach to infer the dynamics of NF-κB kinetics in single cells and cell populations. Using a single system, novel factors modulating NF-κB can be identified and analyzed, providing new possibilities for a wide range of applications from therapeutic discovery and understanding of disease to host-pathogen interactions.
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