Galindo Gabriel, Fixen Gretchen M, Heredia Amelia, Morisaki Tatsuya, Stasevich Timothy J
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
Mol Biol Cell. 2025 Jun 1;36(6):mr6. doi: 10.1091/mbc.E25-02-0091.
Live-cell single-mRNA imaging of translation is inherently challenging, demanding precise optimization of multiple imaging components. To simplify these experiments, we developed All Probes Plasmids (APPs)-a panel of plasmids encoding all the necessary probes for imaging at optimized relative expression levels. APPs incorporate widely used translation tags, fluorescent proteins, and mRNA labeling systems, streamlining both multiplexed imaging and reporter immobilization. By cotransfecting just two plasmids-a reporter and an APP-individual translation sites can be visualized in living cells with high signal-to-noise. We demonstrate how APPs facilitate high-fidelity multicolor translation imaging, long-term single-mRNA tracking, and fluorescence correlation spectroscopy to quantify ribosome kinetics. By lowering technical barriers and enhancing experimental flexibility, APPs provide a versatile platform for advancing single-mRNA translation research in living cells.
活细胞单信使核糖核酸(mRNA)翻译成像本质上具有挑战性,需要对多个成像组件进行精确优化。为了简化这些实验,我们开发了全探针质粒(APPs)——一组质粒,编码用于以优化的相对表达水平进行成像的所有必要探针。APPs整合了广泛使用的翻译标签、荧光蛋白和mRNA标记系统,简化了多重成像和报告基因固定。通过共转染仅两个质粒——一个报告基因和一个APP——单个翻译位点可以在活细胞中以高信噪比可视化。我们展示了APPs如何促进高保真多色翻译成像、长期单mRNA追踪以及荧光相关光谱法来量化核糖体动力学。通过降低技术障碍并增强实验灵活性,APPs为推进活细胞中单mRNA翻译研究提供了一个通用平台。