Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

Détails

Ressource 1Télécharger: BIB_E5E46BFCD092.P001.pdf (3584.17 [Ko])
Etat: Public
Version: Final published version
ID Serval
serval:BIB_E5E46BFCD092
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.
Périodique
PLoS One
Auteur(s)
Detert Oude Weme R.G., Kovács Á.T., de Jong S.J., Veening J.W., Siebring J., Kuipers O.P.
ISSN
1932-6203 (Electronic)
ISSN-L
1932-6203
Statut éditorial
Publié
Date de publication
2015
Volume
10
Numéro
4
Pages
e0123239
Langue
anglais
Résumé
Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.
Mots-clé
Bacillus subtilis/metabolism, Fluorescence Resonance Energy Transfer/methods, Green Fluorescent Proteins/metabolism, Humans, Microscopy, Fluorescence, Molecular Sequence Data
Pubmed
Web of science
Open Access
Oui
Création de la notice
11/10/2016 15:35
Dernière modification de la notice
20/08/2019 16:09
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