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Methods Mol Biol. 2019;2038:131-150. doi: 10.1007/978-1-4939-9674-2_9.

Live-Cell Imaging of mRNP-NPC Interactions in Budding Yeast.

Author information

1
Department of Cell Biology, University of Alberta, Edmonton, Canada.
2
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.
3
Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC, Canada.
4
Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.
5
Faculty of Medicine, Division of Experimental Medicine, McGill University, Montréal, QC, Canada.
6
Department of Cell Biology, University of Alberta, Edmonton, Canada. ben.montpetit@ucdavis.edu.
7
Department of Viticulture and Enology, University of California, Davis, Davis, CA, USA. ben.montpetit@ucdavis.edu.

Abstract

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.

KEYWORDS:

Budding yeast; Fluorescent imaging; Live-cell imaging; NPC; Nuclear pore complex; PP7; S. cerevisiae; Single molecule; Superregistration; mRNP export

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