scholarly journals BRED: bioluminescence energy transfer to dye for monitoring ceramide trafficking in cell

2021 ◽  
Author(s):  
Gita Naseri ◽  
Christoph Arenz
Keyword(s):  
Energy Transfer ◽  
High Efficiency ◽  
Single Cell Analysis ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Analysis ◽  
Bioluminescence Resonance Energy Transfer ◽  
Donor And Acceptor ◽  
Ligand Interactions

Bioluminescence resonance energy transfer (BRET) is a genetically encoded proximity-based tool to study biomolecular interactions. However, conventional BRET is usually restricted to only a few types of interactions like protein-protein or protein-ligand interactions. We here developed a spatially unbiased resonance energy transfer system, so-called BRED - bioluminescence resonance energy transfer to dye. BRED allows transferring energy from a genetically encoded bright human optimized luciferase to a fluorophore-labelled small molecule. The high efficiency of the system allows RET without specific interaction of donor and acceptor. Here, we applied BRED to monitor the trafficking of the signalling lipid ceramide, to the Golgi. This was enabled by an engineered Golgi-resident luciferase, which was used to sense the influx of BODIPY-labeled ceramide into the surrounding membrane. We demonstrated the implementation of the method via flow cytometry, thereby combining the sensitivity of bulk cell methods with the advantages of single-cell analysis. This toolbox enables simple and robust live-cell analysis of inhibitors of CERT-mediated ceramide transport. The design principle of our optogenetic tool can be applied to study intracellular trafficking of metabolites and screen for inhibitors of their key enzymes.

scholarly journals Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Kaku ◽  
Kazunori Sugiura ◽  
Tetsuyuki Entani ◽  
Kenji Osabe ◽  
Takeharu Nagai
Keyword(s):  
Energy Transfer ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Color Change ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bacterial Luciferase ◽  
Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

scholarly journals Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET)

2011 ◽  
Vol 29 (1) ◽  
pp. 119-124 ◽  
Author(s):  
Helen Dacres ◽  
Jian Wang ◽  
Virginia Leitch ◽  
Irene Horne ◽  
Alisha R. Anderson ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer

scholarly journals Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

1998 ◽  
Vol 142 (1) ◽  
pp. 69-84 ◽  
Author(s):  
A.K. Kenworthy ◽  
M. Edidin
Keyword(s):  
Energy Transfer ◽  
Lipid Rafts ◽  
Fluorescence Resonance Energy Transfer ◽  
Mdck Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Microdomains ◽  
Apical Surface ◽  
Donor And Acceptor ◽  
Fluorescence Resonance

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (&lt;100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.

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