The nucleolus has core functions in ribosome biosynthesis, but also acts as a regulatory hub in a plethora of non-canonical processes, including cellular stress. Upon DNA damage, several DNA repair factors shuttle between the nucleolus and the nucleoplasm. Yet, the molecular mechanisms underlying such spatio-temporal protein dynamics remain to be deciphered. Here, we present a novel imaging platform to investigate nucleolar-nucleoplasmic protein shuttling in living cells. For image acquisition, we used a commercially available automated fluorescence microscope and for image analysis, we developed a KNIME workflow with implementation of machine learning-based tools. We validated the method with different nucleolar proteins, i.e., PARP1, TARG1 and APE1, by monitoring their shuttling dynamics upon oxidative stress. As a paradigm, we analyzed PARP1 shuttling upon H2O2 treatment in combination with a range of pharmacological inhibitors in a novel reporter cell line. These experiments revealed that inhibition of SIRT7 results in a loss of nucleolar PARP1 localization. Finally, we unraveled specific differences in PARP1 shuttling dynamics after co-treatment with H2O2 and different clinical PARP inhibitors. Collectively, this work delineates a highly sensitive and versatile bioimaging platform to investigate swift nucleolar-nucleoplasmic protein shuttling in living cells, which can be employed for pharmacological screening and in-depth mechanistic analyses.

中文翻译：

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通过高内涵显微镜和自动图像分析监测活细胞中核仁-核质蛋白的穿梭


核仁在核糖体生物合成中具有核心功能，但也充当许多非规范过程（包括细胞应激）的调节中心。 DNA 损伤后，多种 DNA 修复因子在核仁和核质之间穿梭。然而，这种时空蛋白质动力学背后的分子机制仍有待破译。在这里，我们提出了一种新颖的成像平台来研究活细胞中核仁-核质蛋白的穿梭。对于图像采集，我们使用了市售的自动荧光显微镜；对于图像分析，我们开发了 KNIME 工作流程，并实施了基于机器学习的工具。我们通过监测氧化应激时不同核仁蛋白（即 PARP1、TARG1 和 APE1）的穿梭动态，验证了该方法。作为范例，我们分析了 H2O2 与一系列药理抑制剂联合处理后在新型报告细胞系中的 PARP1 穿梭。这些实验表明，抑制 SIRT7 会导致核仁 PARP1 定位丧失。最后，我们揭示了 H2O2 和不同临床 PARP 抑制剂共同治疗后 PARP1 穿梭动力学的具体差异。总的来说，这项工作描绘了一个高度敏感和多功能的生物成像平台，用于研究活细胞中快速核仁-核质蛋白穿梭，可用于药理学筛选和深入的机制分析。