Single Cell FRET Analysis for the Identification of Optimal FRET-pairs in Bacillus Subtilis Using a Prototype MEM-FLIM System

PLoS One. 2015 Apr 17;10(4):e0123239. doi: 10.1371/journal.pone.0123239. eCollection 2015.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / metabolism*
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Microscopy, Fluorescence
  • Molecular Sequence Data

Substances

  • Green Fluorescent Proteins

Associated data

  • GENBANK/KM009065

Grant support

J.W.V. is supported by the EMBO Young Investigator Programme, a VIDI fellowship (864.12.001) from the Netherlands Organisation for Scientific Research, Earth and Life Sciences (NWO-ALW) and ERC starting grant 337399-PneumoCell. O.P.K. was supported by several STW grants (NWO), two EU FW7 grants: SynPeptide and BachBerry and R.D.O.W. and O.P.K. by a specific SYSMO2 grant from ALW-NWO. Lambert Instruments B.V. provided support in the form of salary for author S.J., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.