Lari Azra, Farzam Farzin, Bensidoun Pierre, Oeffinger Marlene, Zenklusen Daniel, Grunwald David, Montpetit Ben
Department of Cell Biology, University of Alberta, Edmonton, Canada.
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.
Methods Mol Biol. 2019;2038:131-150. doi: 10.1007/978-1-4939-9674-2_9.
Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of interactions between mRNPs and nuclear pore complexes (NPCs) in the yeast S. cerevisiae, including mRNP export. We describe in detail the steps that allow for high-resolution live-cell mRNP imaging and measurement of mRNP interactions with NPCs using simultaneous two-color imaging. Our protocol discusses yeast strain construction, choice of marker proteins to label the nuclear pore complex, as well as imaging conditions that allow high signal-to-noise data acquisition. Moreover, we describe various aspects of postacquisition image analysis for single molecule tracking and image registration allowing for the characterization of mRNP-NPC interactions.
单分子分辨率成像已成为细胞生物学研究中的一项重要工具。基于适体的方法（例如，MS2和PP7）能够检测活细胞中的单个RNA分子，并已被用于研究mRNA代谢的各个方面，包括mRNP核输出。在此，我们概述了一种用于研究酿酒酵母中mRNP与核孔复合体（NPC）之间相互作用（包括mRNP输出）的成像方案。我们详细描述了实现高分辨率活细胞mRNP成像以及使用同步双色成像测量mRNP与NPC相互作用的步骤。我们的方案讨论了酵母菌株构建、用于标记核孔复合体的标记蛋白的选择，以及允许采集高信噪比数据的成像条件。此外，我们描述了用于单分子追踪和图像配准的采集后图像分析的各个方面，以便对mRNP-NPC相互作用进行表征。
