Live-Cell Imaging of mRNP–NPC Interactions in Budding Yeast
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.
Key wordsmRNP export Nuclear pore complex NPC Live-cell imaging Single molecule Budding yeast S. cerevisiae Fluorescent imaging PP7 Superregistration
We would like to acknowledge the laboratories of Drs. Robert Singer and Karsten Weis for reagents and support of previous works related to the methods described here. A.L. was supported by a Natural Sciences and Engineering Research Council Canada Graduate Scholarship; D.Z. is supported by the Canadian Institutes of Health (Project Grant-366682), Fonds de recherche du Québec—Santé (Chercheur-boursier Junior 2), Canada Foundation for Innovation, and the Natural Sciences and Engineering Research Council; D.G. by a National Institute of General Medical Sciences award (5R01GM123541); B.M. and D.G. by a National Institute of General Medical Sciences award (5R01GM124120). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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