Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

  1. Christina K Kim
  2. Kelvin F Cho
  3. Min Woo Kim
  4. Alice Y Ting  Is a corresponding author
  1. Stanford University, United States
  2. Chan Zuckerberg Biohub, United States
5 figures, 2 tables and 1 additional file

Figures

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 …

https://doi.org/10.7554/eLife.43826.002
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. …

https://doi.org/10.7554/eLife.43826.003
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 …

https://doi.org/10.7554/eLife.43826.004
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 …

https://doi.org/10.7554/eLife.43826.007
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 …

https://doi.org/10.7554/eLife.43826.008
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 …

https://doi.org/10.7554/eLife.43826.011
Figure 4—source data 1

Excel spreadsheet containing luminescence values used to generate panel B.

https://doi.org/10.7554/eLife.43826.014
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, …

https://doi.org/10.7554/eLife.43826.012
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 …

https://doi.org/10.7554/eLife.43826.016
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 …

https://doi.org/10.7554/eLife.43826.017
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
DesignationSource or
reference
IdentifiersAdditional
information
Cell line (HEK293T)HEK293TATCCTested negative for mycoplasma.
AntibodyRabbit anti-HACell Signaling TechnologyC29F41:100
AntibodyGoat anti-Rabbit AlexaFluor 647Life TechnologiesA270401:1000
AntibodyMouse anti-FlagSigmaF31651:300
AntibodyGoat anti-Mouse AlexaFluor 568Life TechnologiesA110041:1000
Recombinant
DNA reagent
See Table 1 for all plasmids used or generated in this study.
Commercial
assay or kit
Nano-Glo Live Cell Assay SystemPromega1:100 substrate
Commercial
assay or kit
Bright-Glo Luciferase Assay SystemPromega1:1 substrate
Software,
algorithm
FijiSchindelin et al., 2012
Software,
algorithm
CellSegm toolboxHodneland et al., 2013
Software,
algorithm
MATLAB R2017aMathworks
Table 1
Plasmids used in this study.
https://doi.org/10.7554/eLife.43826.020
NameDescriptionVector-PromoterAddgene
 P1HA-β2AR-NNES-NanoLuc-15aa linker-AsLOV2(V416L)pAAV-CMV125224
 P2gLuc sp-NanoLuc-HA-β2AR-NNES-AsLOV2(V416L)pAAV-CMV125225
 P3mCherry-GSGS linker-Zdk1pAAV-CMV125226
 P4DRD1-NNES-eLOV-TEVcs-Flag-Gal4-V5pAAV-CMV125227
 P5AVPR2-NNES-eLOV-TEVcs-Flag-Gal4-V5pAAV-CMV104844
 P6β2AR-NNES-eLOV-TEVcs-Flag-Gal4-V5pAAV-CMV104841
 P7NanoLuc-15aa linker-βarrestin2-HA-GS linker-TEVppAAV-CMV125228
 P8UAS-CitrinepAAV-UAS104839
 P9βarrestin2-HA-GS linker-TEVppAAV-CMV104845
 P10UAS-FLucpAAV-UAS104840
 P11IgK sp-HA-CD4-10aa linker-CIBN-NNES-NanoLucpAAV-CMV125229
 P12gLuc sp-NanoLuc-HA-oβ2AR-TS-eLOV-TEVcs-Flag-Gal4pLX208-CMV125230
 P13NanoLuc-15aa linker-βarrestin2-HA-GS linker-TEVppLX208-CMV125231
 P14UAS-CitrinepFPGW-UAS125232
 P15gLuc sp-NanoLuc-15aa linker-ICAM-Nrxn3b-HApLX208-CMV125233
 P16gLuc sp-GS linker-HiBit-Flag-oβ2AR-TS-NNES-eLOV-TEVcs-Flag-Gal4-V5pAAV-CMV125234
 P17NanoLuc-15aa linker-βarrestin2-no HA-GS linker-TEVppAAV-CMV125235
 P18β2AR-NNES-NanoLuc-eLOV-TEVcs-Flag-Gal4-V5pAAV-CMV125236

Additional files

Download links