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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Gene Expression Regulation, Fungal
  • Microscopy, Fluorescence*
  • Molecular Imaging / methods*
  • Nuclear Pore / genetics
  • Nuclear Pore / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Single Molecule Imaging / methods*
  • Time Factors

Substances

  • Recombinant Fusion Proteins
  • Ribonucleoproteins
  • Saccharomyces cerevisiae Proteins
  • messenger ribonucleoprotein