Long-term imaging of individual mRNA molecules in living cells.
Long-term imaging of individual mRNA molecules in living cells.
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DOI：
10.1016/j.crmeth.2022.100226
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发表时间：
2022-06-20
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单个mrna的单细胞成像揭示了中心教条的核心机制。然而，大多数方法需要细胞固定或对活细胞应用的灵敏度有限。在这里，我们描述了SunRISER（基于suntag的成像信号富集mRNA报告器），这是一种计算和实验优化的方法，用于明确检测活细胞中的单个mRNA分子。当通过荧光显微镜观察时，sunrise标记的mRNA显示出对背景的强信号和对光漂白的抗性，这两者一起使长期的mRNA成像研究成为可能。使用8倍和10倍茎环阵列的SunRISER变体显示出有效的mRNA检测，同时显着减少对目标mRNA序列的改变。我们对SunRISER进行了表征，观察有丝分裂过程中的mRNA遗传，发现应激源增强了有丝分裂后姐妹细胞的多样性。综上所述，SunRISER可以一瞥活细胞，观察中心法则的各个方面，以及mrna在罕见和动态运输事件中的作用。SunRISER是一种成像活细胞中单个细胞质mRNA的方法，SunRISER标记的mRNA具有高信号强度和抗光漂白性，SunRISER适用于短茎环阵列而不影响mRNA检测，在胁迫下有丝分裂mRNA分配增加姐妹细胞之间的多样性，确定mRNA在罕见事件中的作用和mRNA的动态时空特性需要有效的活细胞成像技术。尽管出现了许多用于增强标记单个mrna的信号强度的工具，但仍然缺乏通过标准显微镜长时间观察mrna的活细胞报告者。因此，我们开发了一种计算和实验优化的方法，称为SunRISER（基于suntag的成像信号富集mRNA报告器），可以在数小时到数天的时间尺度上对单个mRNA进行明确的活细胞检测。在这一期中，Guo等人使用计算和实验方法优化了mRNA标记系统，该系统可以对活细胞中的单个mRNA分子进行长期成像。通过追踪有丝分裂细胞的mRNA遗传，他们发现环境压力促进了母女和姐妹细胞对之间的多样性。
Single-cell imaging of individual mRNAs has revealed core mechanisms of the central dogma. However, most approaches require cell fixation or have limited sensitivity for live-cell applications. Here, we describe SunRISER (SunTag-based reporter for imaging signal-enriched mRNA), a computationally and experimentally optimized approach for unambiguous detection of single mRNA molecules in living cells. When viewed by epifluorescence microscopy, SunRISER-labeled mRNAs show strong signal to background and resistance to photobleaching, which together enable long-term mRNA imaging studies. SunRISER variants, using 8× and 10× stem-loop arrays, demonstrate effective mRNA detection while significantly reducing alterations to target mRNA sequences. We characterize SunRISER to observe mRNA inheritance during mitosis and find that stressors enhance diversity among post-mitotic sister cells. Taken together, SunRISER enables a glimpse into living cells to observe aspects of the central dogma and the role of mRNAs in rare and dynamical trafficking events. SunRISER is an approach for imaging single cytoplasmic mRNAs in living cells SunRISER-labeled mRNAs show high signal intensity and are resistant to photobleaching SunRISER is amenable to short stem-loop arrays without compromising mRNA detection Mitotic mRNA partitioning during stress increases diversity between sister cells Determining the roles of mRNAs in rare events and dynamic spatiotemporal properties of mRNAs requires effective live-cell imaging techniques. Despite the emergence of many tools developed for enhancing signal intensity of labeling single mRNAs, there is still a lack of live-cell reporters to observe mRNAs by standard microscopy over long periods of time. We therefore developed a computationally and experimentally optimized method called SunRISER (SunTag-based reporter for imaging signal-enriched mRNA) that enables unambiguous live-cell detection of single mRNAs over timescales of hours to days. In this issue, Guo et al. use computational and experimental approaches to optimize an mRNA-labeling system that enables long-term imaging of single mRNA molecules in living cells. By tracking mRNA inheritance in cells undergoing mitosis, they show that environmental stresses promote diversity between mother-daughter and sister-cell pairs.