Live-Cell Imaging of mRNP-NPC Interactions in Budding Yeast.
Active Transport, Cell Nucleus
Gene Expression Regulation, Fungal
Microscopy, Fluorescence
Molecular Imaging
/ methods
Nuclear Pore
/ genetics
Recombinant Fusion Proteins
/ genetics
Ribonucleoproteins
/ genetics
Saccharomyces cerevisiae
/ genetics
Saccharomyces cerevisiae Proteins
/ genetics
Single Molecule Imaging
/ methods
Time Factors
Budding yeast
Fluorescent imaging
Live-cell imaging
NPC
Nuclear pore complex
PP7
S. cerevisiae
Single molecule
Superregistration
mRNP export
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2019
2019
Historique:
entrez:
14
8
2019
pubmed:
14
8
2019
medline:
9
4
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31407282
doi: 10.1007/978-1-4939-9674-2_9
pmc: PMC7392168
mid: NIHMS1608774
doi:
Substances chimiques
Recombinant Fusion Proteins
0
Ribonucleoproteins
0
Saccharomyces cerevisiae Proteins
0
messenger ribonucleoprotein
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
131-150Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM124120
Pays : United States
Organisme : NIDA NIH HHS
ID : U01 DA047733
Pays : United States
Organisme : NIBIB NIH HHS
ID : U01 EB021238
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM123541
Pays : United States
Organisme : CIHR
ID : 366682
Pays : Canada
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