Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions
- Stanford University, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1
Motivation and design for SPARK2.
(A) Logic diagram for single ‘AND’ gate used in first-generation SPARK1 (Kim et al., 2017). Transcription of the reporter gene requires both light (to open the LOV domain) AND a protein-protein …
Figure 2 with 1 supplement
Efficient and proximity-dependent NanoLuc-LOV BRET detected using LOVTRAP.
(A) Schematic of NanoLuc fused to membrane-localized protein β2AR and asLOV2 in LOVTRAP. When asLOV2 is activated by NanoLuc (or blue light), mCherry-Zdk1 dissociates from the C-terminus of asLOV2. …
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Figure 2—source data 1
Excel spreadsheet containing fluorescence intensity values used to generate panels C-D and G-H.
- https://doi.org/10.7554/eLife.43826.006
Figure 2—figure supplement 1
Quantification and controls for LOVTRAP detection of NanoLuc-LOV activation.
(A) HEK293T cells were transfected as in Figure 2A. Example sequential immunofluorescence images of mCherry-Zdk1 during baseline (Dark), 10 µM furimazine, and blue light for the intracellular …
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Figure 2—figure supplement 1—source data 1
Excel spreadsheet containing fluorescence intensity and luminescence values used to generate panels B-D.
- https://doi.org/10.7554/eLife.43826.005
Figure 3 with 1 supplement
Reduced background during transcriptional read-out of PPIs with a nested ‘AND’ gate in SPARK2.
(A) Schematic of NanoLuc fused to arrestin-TEVp, co-expressed with GPCR-LOV-TEVcs-Gal4 and UAS-Citrine. In the dark and in the absence of the GPCR’s agonist, the TEVcs is caged by the LOV domain and …
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Figure 3—source data 1
Excel spreadsheet containing fluorescence ratio intensity values used to generate panels D, G, and H.
- https://doi.org/10.7554/eLife.43826.010
Figure 3—figure supplement 1
Direct comparison of SPARK1 versus SPARK2 and raw fluorescence intensities for SPARK2 characterization.
(A) HEK293T cells were transfected with either β2AR SPARK1 (arrestin-TEVp) or SPARK2 (NanoLuc-arrestin-TEVp) and UAS-Citrine. The same transmembrane construct was used in both conditions …
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Figure 3—figure supplement 1—source data 1
Excel spreadsheet containing fluorescence intensity ratio values used to generate panel B.
- https://doi.org/10.7554/eLife.43826.009
Figure 4 with 1 supplement
SPARK2 is compatible with an orthogonal luciferase reporter to enable high-throughput drug-screens for GPCR activation.
(A) Schematic of the general timeline for SPARK2 assays using an orthogonal luciferase reporter such as FLuc. For simplicity, only the luciferases and LOV domain are illustrated (unfilled …
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Figure 4—source data 1
Excel spreadsheet containing luminescence values used to generate panel B.
- https://doi.org/10.7554/eLife.43826.014
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Figure 4—source data 2
Excel spreadsheet containing SPARK2 drug screen compounds and mean fold-changes (n = 2 biological replicates) to generate panels C-E.
- https://doi.org/10.7554/eLife.43826.015
Figure 4—figure supplement 1
Quantification and controls for proximity-dependence of SPARK2.
(A) Schematic for testing whether NanoLuc activation of eLOV is proximity-dependent. NanoLuc was fused either to arrestin-TEVp (left, Complexed), or expressed at the membrane as CD4-NanoLuc (right, …
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Figure 4—figure supplement 1—source data 1
Excel spreadsheet containing luminescence values used to generate panels B-D.
- https://doi.org/10.7554/eLife.43826.013
Figure 5 with 1 supplement
Detection of cell-cell contacts using NanoLuc-oβ2AR activation with SPARK2.
(A) Schematic of an extracellular NanoLuc fused to oβ2AR-eLOV-TEVcs-Gal4, co-expressed with NanoLuc-arrestin-TEVp and UAS-Citrine. (B) Citrine expression in HEK293T cells infected with lentiviruses …
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Figure 5—source data 1
Excel spreadsheet containing cell count and fluorescence intensity ratio values used to generate panels C and G.
- https://doi.org/10.7554/eLife.43826.019
Figure 5—figure supplement 1
Quantification and controls for detection of cell-cell contacts using NanoLuc-oβ2AR activation with SPARK2.
(A) HEK293T cells were infected with lentiviruses as in Figure 5A. Immunofluorescence images of Citrine expression were taken ~24 hr following 10 min of blue light to activate …
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Figure 5—figure supplement 1—source data 1
Excel spreadsheet containing cell counts and fluorescence intensity ratio values used to generate panels B and D-E.
- https://doi.org/10.7554/eLife.43826.018
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (HEK293T) | HEK293T | ATCC | Tested negative for mycoplasma. | |
| Antibody | Rabbit anti-HA | Cell Signaling Technology | C29F4 | 1:100 |
| Antibody | Goat anti-Rabbit AlexaFluor 647 | Life Technologies | A27040 | 1:1000 |
| Antibody | Mouse anti-Flag | Sigma | F3165 | 1:300 |
| Antibody | Goat anti-Mouse AlexaFluor 568 | Life Technologies | A11004 | 1:1000 |
| Recombinant DNA reagent | See Table 1 for all plasmids used or generated in this study. | |||
| Commercial assay or kit | Nano-Glo Live Cell Assay System | Promega | 1:100 substrate | |
| Commercial assay or kit | Bright-Glo Luciferase Assay System | Promega | 1:1 substrate | |
| Software, algorithm | Fiji | Schindelin et al., 2012 | ||
| Software, algorithm | CellSegm toolbox | Hodneland et al., 2013 | ||
| Software, algorithm | MATLAB R2017a | Mathworks |
Table 1
Plasmids used in this study.
https://doi.org/10.7554/eLife.43826.020| Name | Description | Vector-Promoter | Addgene |
|---|---|---|---|
| P1 | HA-β2AR-NNES-NanoLuc-15aa linker-AsLOV2(V416L) | pAAV-CMV | 125224 |
| P2 | gLuc sp-NanoLuc-HA-β2AR-NNES-AsLOV2(V416L) | pAAV-CMV | 125225 |
| P3 | mCherry-GSGS linker-Zdk1 | pAAV-CMV | 125226 |
| P4 | DRD1-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 125227 |
| P5 | AVPR2-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 104844 |
| P6 | β2AR-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 104841 |
| P7 | NanoLuc-15aa linker-βarrestin2-HA-GS linker-TEVp | pAAV-CMV | 125228 |
| P8 | UAS-Citrine | pAAV-UAS | 104839 |
| P9 | βarrestin2-HA-GS linker-TEVp | pAAV-CMV | 104845 |
| P10 | UAS-FLuc | pAAV-UAS | 104840 |
| P11 | IgK sp-HA-CD4-10aa linker-CIBN-NNES-NanoLuc | pAAV-CMV | 125229 |
| P12 | gLuc sp-NanoLuc-HA-oβ2AR-TS-eLOV-TEVcs-Flag-Gal4 | pLX208-CMV | 125230 |
| P13 | NanoLuc-15aa linker-βarrestin2-HA-GS linker-TEVp | pLX208-CMV | 125231 |
| P14 | UAS-Citrine | pFPGW-UAS | 125232 |
| P15 | gLuc sp-NanoLuc-15aa linker-ICAM-Nrxn3b-HA | pLX208-CMV | 125233 |
| P16 | gLuc sp-GS linker-HiBit-Flag-oβ2AR-TS-NNES-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 125234 |
| P17 | NanoLuc-15aa linker-βarrestin2-no HA-GS linker-TEVp | pAAV-CMV | 125235 |
| P18 | β2AR-NNES-NanoLuc-eLOV-TEVcs-Flag-Gal4-V5 | pAAV-CMV | 125236 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.43826.021
