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

Detert Oude Weme RG, Kovács ÁT, de Jong SJ, Veening JW, Siebring J, Kuipers OP - PLoS ONE (2015)

Bottom Line: 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.This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.

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.

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The FRET efficiency, Ea, was determined over time with a fluorescence microscopy time-lapse experiment.The covalently bound GFP-tagRFP, e.g. no TEV-protease, results in a high FRET efficiency (red line) and when the GFP-tagRFP is uncoupled by inducing the TEV-protease encoding gene, it results in a low FRET efficiency (black line). Error bars show the standard deviation of three replicate experiments. At least 50 single cells were analyzed at each time point.
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pone.0123239.g004: The FRET efficiency, Ea, was determined over time with a fluorescence microscopy time-lapse experiment.The covalently bound GFP-tagRFP, e.g. no TEV-protease, results in a high FRET efficiency (red line) and when the GFP-tagRFP is uncoupled by inducing the TEV-protease encoding gene, it results in a low FRET efficiency (black line). Error bars show the standard deviation of three replicate experiments. At least 50 single cells were analyzed at each time point.

Mentions: This study focusses on finding appropriate FRET-pairs for studying temporal behavior of protein-protein interactions. The GFP-tagRFP FRET pair was used in a time-lapse experiment to determine if the FRET efficiency is stable over time. Both covalently bound and TEV-protease treated GFP-tagRFP produce a constant FRET efficiency (Fig 4). FRET-efficiency altering processes like unequal protein degradation was hereby excluded. Any FRET-efficiency dynamics found in future protein-protein interaction experiments can be attributed to the given protein-protein interactions.


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

Detert Oude Weme RG, Kovács ÁT, de Jong SJ, Veening JW, Siebring J, Kuipers OP - PLoS ONE (2015)

The FRET efficiency, Ea, was determined over time with a fluorescence microscopy time-lapse experiment.The covalently bound GFP-tagRFP, e.g. no TEV-protease, results in a high FRET efficiency (red line) and when the GFP-tagRFP is uncoupled by inducing the TEV-protease encoding gene, it results in a low FRET efficiency (black line). Error bars show the standard deviation of three replicate experiments. At least 50 single cells were analyzed at each time point.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401445&req=5

pone.0123239.g004: The FRET efficiency, Ea, was determined over time with a fluorescence microscopy time-lapse experiment.The covalently bound GFP-tagRFP, e.g. no TEV-protease, results in a high FRET efficiency (red line) and when the GFP-tagRFP is uncoupled by inducing the TEV-protease encoding gene, it results in a low FRET efficiency (black line). Error bars show the standard deviation of three replicate experiments. At least 50 single cells were analyzed at each time point.
Mentions: This study focusses on finding appropriate FRET-pairs for studying temporal behavior of protein-protein interactions. The GFP-tagRFP FRET pair was used in a time-lapse experiment to determine if the FRET efficiency is stable over time. Both covalently bound and TEV-protease treated GFP-tagRFP produce a constant FRET efficiency (Fig 4). FRET-efficiency altering processes like unequal protein degradation was hereby excluded. Any FRET-efficiency dynamics found in future protein-protein interaction experiments can be attributed to the given protein-protein interactions.

Bottom Line: 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.This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, 9747 AG Groningen, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus