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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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Single cells FLIM measurements.(A1) B. subtilis cells are shown where the GFP-tagRFP fluorophores are linked. (B1) B. subtilis cells are presented where the GFP and tagRFP fluorophores are cleaved apart. (C1) B. subtilis cells where GFP-tagRFP fluorophores are linked are mixed in a 1:1 ratio with B. subtilis cells where the GFP-tagRFP fluorophores are cleaved apart; resulting in a mix of cells with either short GFP fluorescence lifetime due to quenching by tagRFP or long GFP fluorescence lifetime. Visualization of cells in A1, B1, C1 was done with a Look-Up-Table from LI-FLIM. A2, B2 and C2 present the same cells, but now a Matlab script was used to categorize the cells into two categories: cells with short GFP lifetimes are shown in cyan and cells with long GFP lifetimes are shown in magenta. (D) fluorescence lifetime based histogram of the cells described in A2-C2, black, cyan, magenta and dotted lines present GFP_only, linked fluorophores, cleaved fluorophores and a mix of the two populations, respectively. Scale bar is 5 μm.
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pone.0123239.g005: Single cells FLIM measurements.(A1) B. subtilis cells are shown where the GFP-tagRFP fluorophores are linked. (B1) B. subtilis cells are presented where the GFP and tagRFP fluorophores are cleaved apart. (C1) B. subtilis cells where GFP-tagRFP fluorophores are linked are mixed in a 1:1 ratio with B. subtilis cells where the GFP-tagRFP fluorophores are cleaved apart; resulting in a mix of cells with either short GFP fluorescence lifetime due to quenching by tagRFP or long GFP fluorescence lifetime. Visualization of cells in A1, B1, C1 was done with a Look-Up-Table from LI-FLIM. A2, B2 and C2 present the same cells, but now a Matlab script was used to categorize the cells into two categories: cells with short GFP lifetimes are shown in cyan and cells with long GFP lifetimes are shown in magenta. (D) fluorescence lifetime based histogram of the cells described in A2-C2, black, cyan, magenta and dotted lines present GFP_only, linked fluorophores, cleaved fluorophores and a mix of the two populations, respectively. Scale bar is 5 μm.

Mentions: Cells containing GFP-tagRFP were used to study the usability of the prototype MEM-FLIM system for FRET-FLIM measurements at the single bacterial cell level (Fig 5). In the top part of Fig 5 cells with coupled fluorophores, cells with uncoupled fluorophores or a mix of cells with coupled and uncoupled fluorophores were false-colored with a look-up-table from the LI-FLIM software. Using a Matlab script for automated cell sorting, these cells were categorized into two groups based on fluorescence lifetime values; cells with short lifetime were false-colored cyan and with long lifetime were false-colored magenta; threshold value was set to 2.3 ns (Fig 5A2–5C2). This script was also used to make a fluorescence lifetime based histogram (Fig 5D). The histogram confirms that the cells from Fig 5C2 contained cells with short and long fluorescence lifetime. This showed that the MEM-FLIM prototype allows single bacterial cell FLIM and can resolve inter-individual fluorescence lifetimes.


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)

Single cells FLIM measurements.(A1) B. subtilis cells are shown where the GFP-tagRFP fluorophores are linked. (B1) B. subtilis cells are presented where the GFP and tagRFP fluorophores are cleaved apart. (C1) B. subtilis cells where GFP-tagRFP fluorophores are linked are mixed in a 1:1 ratio with B. subtilis cells where the GFP-tagRFP fluorophores are cleaved apart; resulting in a mix of cells with either short GFP fluorescence lifetime due to quenching by tagRFP or long GFP fluorescence lifetime. Visualization of cells in A1, B1, C1 was done with a Look-Up-Table from LI-FLIM. A2, B2 and C2 present the same cells, but now a Matlab script was used to categorize the cells into two categories: cells with short GFP lifetimes are shown in cyan and cells with long GFP lifetimes are shown in magenta. (D) fluorescence lifetime based histogram of the cells described in A2-C2, black, cyan, magenta and dotted lines present GFP_only, linked fluorophores, cleaved fluorophores and a mix of the two populations, respectively. Scale bar is 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123239.g005: Single cells FLIM measurements.(A1) B. subtilis cells are shown where the GFP-tagRFP fluorophores are linked. (B1) B. subtilis cells are presented where the GFP and tagRFP fluorophores are cleaved apart. (C1) B. subtilis cells where GFP-tagRFP fluorophores are linked are mixed in a 1:1 ratio with B. subtilis cells where the GFP-tagRFP fluorophores are cleaved apart; resulting in a mix of cells with either short GFP fluorescence lifetime due to quenching by tagRFP or long GFP fluorescence lifetime. Visualization of cells in A1, B1, C1 was done with a Look-Up-Table from LI-FLIM. A2, B2 and C2 present the same cells, but now a Matlab script was used to categorize the cells into two categories: cells with short GFP lifetimes are shown in cyan and cells with long GFP lifetimes are shown in magenta. (D) fluorescence lifetime based histogram of the cells described in A2-C2, black, cyan, magenta and dotted lines present GFP_only, linked fluorophores, cleaved fluorophores and a mix of the two populations, respectively. Scale bar is 5 μm.
Mentions: Cells containing GFP-tagRFP were used to study the usability of the prototype MEM-FLIM system for FRET-FLIM measurements at the single bacterial cell level (Fig 5). In the top part of Fig 5 cells with coupled fluorophores, cells with uncoupled fluorophores or a mix of cells with coupled and uncoupled fluorophores were false-colored with a look-up-table from the LI-FLIM software. Using a Matlab script for automated cell sorting, these cells were categorized into two groups based on fluorescence lifetime values; cells with short lifetime were false-colored cyan and with long lifetime were false-colored magenta; threshold value was set to 2.3 ns (Fig 5A2–5C2). This script was also used to make a fluorescence lifetime based histogram (Fig 5D). The histogram confirms that the cells from Fig 5C2 contained cells with short and long fluorescence lifetime. This showed that the MEM-FLIM prototype allows single bacterial cell FLIM and can resolve inter-individual fluorescence lifetimes.

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