Sahota Annie, Paulose Nadappuram Binoy, Kwan Zoe, Lesept Flavie, Howden Jack H, Claxton Suzanne, Kittler Josef T, Devine Michael J, Edel Joshua B, Ivanov Aleksandar P
Department of Chemistry, Imperial College London, Molecular Science Research Hub, London W12 0BZ, United Kingdom.
Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1BX, United Kingdom.
ACS Nano. 2025 May 20;19(19):18522-18533. doi: 10.1021/acsnano.5c02056. Epub 2025 May 6.
Emerging techniques for mapping mRNAs within the subcellular compartments of live cells hold great promise for advancing our understanding of the spatial distribution of transcripts and enabling the study of single-cell dynamics in health and disease. This is particularly critical for polarized cells, such as neurons, where mRNA compartmentalization is essential for regulating gene expression, and defects in these localization mechanisms are linked to numerous neurological disorders. However, many subcellular analysis techniques require a compromise between subcellular precision, live-cell measurements, and nondestructive access to single cells in their native microenvironment. To overcome these challenges, we employ a single-cell technology that we have recently developed, the nanotweezer, which features a nanoscale footprint (∼100 nm), avoids cytoplasmic fluid aspiration, and enables rapid RNA isolation from living cells with minimal invasiveness. Using this tool, we investigate single-cell mRNA compartmentalization in the soma and dendrites of hippocampal neurons at different stages of neuronal development. By combining precise targeting with sequential sampling, we track changes in mRNA abundance at dendritic spine regions of the same neuron, both before and after stimulation. This minimally invasive approach enables time-resolved, subcellular gene expression profiling of the same single cell. This could provide critical insights into polarized cells and advance our understanding of biological processes and complex diseases.
用于绘制活细胞亚细胞区室中mRNA的新兴技术，在推进我们对转录本空间分布的理解以及促进对健康和疾病中单细胞动态的研究方面具有巨大潜力。这对于极化细胞（如神经元）尤为关键，在神经元中mRNA的区室化对于调节基因表达至关重要，并且这些定位机制的缺陷与多种神经疾病相关。然而，许多亚细胞分析技术需要在亚细胞精度、活细胞测量以及在其天然微环境中对单细胞的非破坏性访问之间做出妥协。为了克服这些挑战，我们采用了一种我们最近开发的单细胞技术——纳米镊子，它具有纳米级足迹（约100纳米），避免了细胞质液体抽吸，并能够以最小的侵入性从活细胞中快速分离RNA。使用这个工具，我们研究了神经元发育不同阶段海马神经元胞体和树突中的单细胞mRNA区室化。通过将精确靶向与顺序采样相结合，我们跟踪了同一神经元树突棘区域在刺激前后mRNA丰度的变化。这种微创方法能够对同一单细胞进行时间分辨的亚细胞基因表达谱分析。这可以为极化细胞提供关键见解，并推进我们对生物过程和复杂疾病的理解。
