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

  • Azra Lari
  • Farzin Farzam
  • Pierre Bensidoun
  • Marlene Oeffinger
  • Daniel Zenklusen
  • David Grunwald
  • Ben MontpetitEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)


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 words

mRNP 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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Azra Lari
    • 1
  • Farzin Farzam
    • 2
  • Pierre Bensidoun
    • 3
    • 4
  • Marlene Oeffinger
    • 3
    • 4
    • 5
  • Daniel Zenklusen
    • 3
  • David Grunwald
    • 2
  • Ben Montpetit
    • 1
    • 6
    Email author
  1. 1.Department of Cell BiologyUniversity of AlbertaEdmontonCanada
  2. 2.RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterUSA
  3. 3.Département de Biochimie et Médecine MoléculaireUniversité de MontréalMontréalCanada
  4. 4.Institut de Recherches Cliniques de MontréalMontréalCanada
  5. 5.Faculty of Medicine, Division of Experimental MedicineMcGill UniversityMontréalCanada
  6. 6.Department of Viticulture and EnologyUniversity of California, DavisDavisUSA

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