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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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Fluorescence intensities of single cells for the FRET-pair GFP-tagRFP.Microscope excitation and emission filter settings are shown between brackets (d = donor, a = acceptor). For donor the filters (excitation, emission) were: GFP, GFP; for acceptor: mCherry, mCherry; and for FRET the filters were: GFP, mCherry. In all cases a GFP/mCherry polychroic mirror was used (400–470, 490–570, 580–630 and 640–730 nm range). A and B are cells where only donor fluorophore is present (GFP). C, D and D2 are cells where only acceptor fluorophore is present (tagRFP). E, F and G (upper panel) are cells where donor-acceptor fluorophore (GFP-tagRFP) are coupled and TEV-protease is not induced. E, F and G (lower panel) are cells where donor-acceptor (GFP-tagRFP) are uncoupled by induction of TEV-protease. The same signal scaling is used for all images. Note that the signals are false colored (GFP: green, tagRFP: red). Scale bar is 5 μm.
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pone.0123239.g003: Fluorescence intensities of single cells for the FRET-pair GFP-tagRFP.Microscope excitation and emission filter settings are shown between brackets (d = donor, a = acceptor). For donor the filters (excitation, emission) were: GFP, GFP; for acceptor: mCherry, mCherry; and for FRET the filters were: GFP, mCherry. In all cases a GFP/mCherry polychroic mirror was used (400–470, 490–570, 580–630 and 640–730 nm range). A and B are cells where only donor fluorophore is present (GFP). C, D and D2 are cells where only acceptor fluorophore is present (tagRFP). E, F and G (upper panel) are cells where donor-acceptor fluorophore (GFP-tagRFP) are coupled and TEV-protease is not induced. E, F and G (lower panel) are cells where donor-acceptor (GFP-tagRFP) are uncoupled by induction of TEV-protease. The same signal scaling is used for all images. Note that the signals are false colored (GFP: green, tagRFP: red). Scale bar is 5 μm.

Mentions: The process of data acquisition and analysis to calculate the FRET efficiency is shown with single cell images of the FRET pair GFP-tagRFP (Fig 3 and Table 4). First, cells with only donor and only acceptor were imaged under the microscope in three channels (Fig 3A–3D2). Next, cells with donor and acceptor (the FRET pair) were imaged in the same three channels both with and without induction of TEV-protease (Fig 3E–3G). Note the significant decrease in acceptor fluorescence (Fig 3F) in the presence of TEV-protease. The contributions of donor emission in the acceptor channel (Fig 3B) and the excitation of the acceptor by donor excitation (Fig 3C) were very small, but nevertheless these contributions need to be taken into account, because this light in the FRET channel is not due to sensitized emission of the acceptor. After measuring the fluorescence intensities from all cells (Fig 3) the FRET efficiency measured via sensitized emission was calculated with Eq 6.


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)

Fluorescence intensities of single cells for the FRET-pair GFP-tagRFP.Microscope excitation and emission filter settings are shown between brackets (d = donor, a = acceptor). For donor the filters (excitation, emission) were: GFP, GFP; for acceptor: mCherry, mCherry; and for FRET the filters were: GFP, mCherry. In all cases a GFP/mCherry polychroic mirror was used (400–470, 490–570, 580–630 and 640–730 nm range). A and B are cells where only donor fluorophore is present (GFP). C, D and D2 are cells where only acceptor fluorophore is present (tagRFP). E, F and G (upper panel) are cells where donor-acceptor fluorophore (GFP-tagRFP) are coupled and TEV-protease is not induced. E, F and G (lower panel) are cells where donor-acceptor (GFP-tagRFP) are uncoupled by induction of TEV-protease. The same signal scaling is used for all images. Note that the signals are false colored (GFP: green, tagRFP: red). Scale bar is 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123239.g003: Fluorescence intensities of single cells for the FRET-pair GFP-tagRFP.Microscope excitation and emission filter settings are shown between brackets (d = donor, a = acceptor). For donor the filters (excitation, emission) were: GFP, GFP; for acceptor: mCherry, mCherry; and for FRET the filters were: GFP, mCherry. In all cases a GFP/mCherry polychroic mirror was used (400–470, 490–570, 580–630 and 640–730 nm range). A and B are cells where only donor fluorophore is present (GFP). C, D and D2 are cells where only acceptor fluorophore is present (tagRFP). E, F and G (upper panel) are cells where donor-acceptor fluorophore (GFP-tagRFP) are coupled and TEV-protease is not induced. E, F and G (lower panel) are cells where donor-acceptor (GFP-tagRFP) are uncoupled by induction of TEV-protease. The same signal scaling is used for all images. Note that the signals are false colored (GFP: green, tagRFP: red). Scale bar is 5 μm.
Mentions: The process of data acquisition and analysis to calculate the FRET efficiency is shown with single cell images of the FRET pair GFP-tagRFP (Fig 3 and Table 4). First, cells with only donor and only acceptor were imaged under the microscope in three channels (Fig 3A–3D2). Next, cells with donor and acceptor (the FRET pair) were imaged in the same three channels both with and without induction of TEV-protease (Fig 3E–3G). Note the significant decrease in acceptor fluorescence (Fig 3F) in the presence of TEV-protease. The contributions of donor emission in the acceptor channel (Fig 3B) and the excitation of the acceptor by donor excitation (Fig 3C) were very small, but nevertheless these contributions need to be taken into account, because this light in the FRET channel is not due to sensitized emission of the acceptor. After measuring the fluorescence intensities from all cells (Fig 3) the FRET efficiency measured via sensitized emission was calculated with Eq 6.

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