Single-color, ratiometric biosensors for detecting signaling activities in live cells
- University of California, San Diego, United States
- Johns Hopkins University, United States
- Johns Hopkins University School of Medicine, United States
- University of Maryland Baltimore, United States
Figures
Figure 1 with 8 supplements
Design and characterization of FLARE AKAR.
(a) Schematic of a kinase activity FLARE (b) Diagram illustrating domain structure of FLARE-AKAR (top). Time-course of mean fluorescence anisotropy of Venus-cp172Venus FLARE-AKAR wild type (blue, N =…
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Figure 1—source data 1
FLARE AKAR characterization.
(a) Sheet 1, Figure 1b Time Course. Time course for mVenus-cp17Venus FLARE AKAR, wild type and kinase-inactive threonine-alanine (TA) mutant. (b) Sheet 2, Figure 1b Summary: Comparison of magnitude of responses for individual cells for Venus-cp172Venus FLARE AKAR, wild type and kinase-inactive threonine-alanine (TA) mutant, as well as statistics comparing the two groups. (c) Sheet 3, Figure 1c. Summary of magnitude of responses for various color variants of FLARE AKAR after stimulation with forskolin and IBMX.
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Figure 1—figure supplement 1
Anisotropy vs. intramolecular distance.
Anisotropy measurements of monomer, as well as tandem dimers separated by either a long (44 amino acid) or a short (10 amino acid) linker, of (a) mCerulean3, (b) mVenus, and (c) mCherry. The mean …
Figure 1—figure supplement 2
Individual and average traces for FLARE-AKAR panel.
Individual and average traces for FLARE-AKAR variants, including (a) Venus-cp172Venus AKAR (N = 44), (b) Venus-cp172Venus FLARE-AKAR T/A mutant (N = 38), (c) Venus-Venus FLARE AKAR (N = 32), (d) …
Figure 1—figure supplement 3
Anisotropy change vs expression level.
Scatter plots showing the relationship between the magnitude of the anisotropy change after treatment with 50 μM forskolin and 100 μM IBMX and the initial intensity for (a) Venus-cp172Venus FLARE …
Figure 1—figure supplement 4
Venus-cp172Venus FLARE-AKAR isoproterenol dose response.
HEK293T cells were transiently transfected with Venus-cp172Venus FLARE-AKAR, and stimulated with varying doses of isoproterenol, followed by maximal stimulation with 50 μM forskolin and 100 μM IBMX. …
Figure 1—figure supplement 5
Characterization of the chromophore-dead FLARE AKAR mutant.
(a) Comparison of Venus-cp172Venus FLARE AKAR responses of the wild type sensor and a mutant sensor in which the chromophore of the C-terminal cp172Venus was mutated from GYG to GGG (N = 53), …
Figure 1—figure supplement 6
Direct comparison of mCherry-mCherry FLARE-AKAR and heteroFRET AKAR4.
mCherry-mCherry FLARE-AKAR and AKAR4 were co-expressed in HEK293T cells, which were stimulated with 50 μM forskolin and 100 μM IBMX at t = 0 min (N = 74). (a) Average anisotropy traces for …
Figure 1—figure supplement 7
Subcellular targeted FLARE-AKARs.
Average anisotropy traces of Venus-cp172Venus FLARE AKAR subcellularly targeted to (a) the plasma membrane with the Lyn-kinase targeting motif (N = 77) (b) the mitochondrial membrane with the …
Figure 1—figure supplement 8
Differential PKA activity kinetics in the cytosol and the nucleus.
(a) Comparison of Venus-cp172Venus FLARE AKAR anisotropy changes in the cytosol and the nucleus in HeLa cells treated with 50 μM forskolin and 100 μM IBMX (N = 19). Dashed lines above and below the …
Figure 2 with 4 supplements
A panel of kinase activity and activation biosensors.
(a) Domain structure of FLARE-EKAR-EV (above). Time-course of mean fluorescence anisotropy of Venus-cp172Venus FLARE-EKAR-EV WT (blue, N = 13) and kinase-insensitive mutant (red, N = 16) expressed …
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Figure 2—source data 1
FLARE kinase biosensor panel.
(a) Sheet 1, Figure 2a. Time course for Venus-cp172Venus FLARE EKAR, both wild type and kinase-inactive (TA) mutant. (b) Sheet 2, Figure 2a. Comparison of magnitudes of anisotropy changes for Venus-cp172Venus FLARE EKAR, both wild type and kinase-inactive (TA) mutant, of upon EGF addition, and relevant statistical tests to compare the two variants. (c) Sheet 3, Figure 2—figure supplement 1c. Changes of magnitudes of anisotropy change for various FLARE EKAR variants upon EGF addition. (d) Sheet 4, Figure 2b. Time course for Venus-cp172Venus FLARE CKAR, both wild type and kinase-inactive (TA) mutant. (e) Sheet 5, Figure 2b. Comparison of magnitudes of anisotropy changes for Venus-cp172Venus FLARE EKAR, both wild type and kinase-inactive (TA) mutant, of upon EGF addition, and relevant statistical tests to compare the two variants. (f) Sheet 6, Figure 2—figure supplement 2. Time courses for CKAR1 and CKAR2. (g) Sheet 7, Figure 2—figure supplement 3c. Changes of magnitudes of anisotropy change for various FLARE CKAR variants upon PMA addition. (h) Sheet 8, Figure 2c. Time course for FLARE MLCK, with either addition of KCl and vehicle only control. (i) Sheet 9, Figure 2c. Summary for magnitude of responses for FLARE MLCK.
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Figure 2—figure supplement 1
FLARE-EKAR characterization.
(a) Individual (gray) and average (red) cell traces for Venus-cp172Venus FLARE EKAR (N = 13), expressed in HEK293T cells and treated with 100 ng/mL epidermal growth factor (EGF) at t = 0 min and the …
Figure 2—figure supplement 2
Characterization of the CKAR2 hetero-FRET biosensor.
(a) Comparison of CKAR1 and CKAR2 domain structures. (b) Comparison of FRET responses of CKAR1 and CKAR2 expressed in HeLa cells to Protein Kinase C (PKC) stimulation with 50 ng/mL phorbol …
Figure 2—figure supplement 3
FLARE CKAR characterization.
(a) Individual (gray) and average (red) cell traces for Venus-cp172Venus FLARE CKAR (N = 26), expressed in HEK293T cells treated with 50 ng/mL PMA at t = 0 min. Dotted lines above and below the …
Figure 2—figure supplement 4
FLARE MLCK characterization.
(a) Individual (gray) and average (red) cell traces for FLARE MLCK (N = 13) with KCl added at t = 0 min (N = 13). (b) Individual (gray) and average (red) cell traces for FLARE-MLCK with vehicle only …
Figure 3 with 4 supplements
Design and characterization of FLARE second messenger biosensors.
(a) Schematic of FLARE ICUE cAMP biosensor. (b) Domain structure of FLARE-Cameleon (top). Time-course of mean fluorescence anisotropy of Venus-cp172Venus FLARE-Cameleon (N = 10) with addition of 1 …
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Figure 3—source data 1
FLARE second messenger biosensor panel.
(a) Sheet 1, Figure 3b. Time course for Venus-cp172Venus FLARE Cameleon. (b) Sheet 2, Figure 3b. Summary of magnitude of responses for Venus-cp172Venus FLARE Cameleon upon addition of calcium chloride and ionomycin. (c) Sheet 3, Figure 3—figure supplement 1b. Summary of magnitudes of responses for various FLARE Cameleon variants upon addition of calcium chloride and ionomycin. (d) Sheet 4, Figure 3—figure supplement 2. In vitro calibration of Venus-cp172Venus FLARE Cameleon, both raw data and sigmoidal curve fits. (e) Sheet 5, Figure 3—figure supplement 3. Summary of magnitude of anisotropy changes for CFP FLARE D1ER upon addition of ionomycin and three different doses of calcium. (f) Sheet 6, Figure 3c. Time course for Venus-cp172Venus FLARE ICUE. (g) Sheet 7, Figure 3c Summary of magnitudes of changes in anisotropy for Venus-cp172Venus FLARE ICUE upon addition of forskolin and IBMX.
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Figure 3—figure supplement 1
Characterization of FLARE Cameleon.
(a) Individual (gray) and average (red) cell traces for Venus-cp172 FLARE Cameleon (N = 10). (b) A summary of anisotropy changes for various color-variants of FLARE-Cameleon in HEK293T cells upon …
Figure 3—figure supplement 2
In vitro calibration of purified Venus-cp172 FLARE-Cameleon.
Anisotropy vs. calcium concentration calibration curves of purified Venus-cp172Venus Cameleon for (a) 25°C (b) 32°C, and (c) 37°C.
Figure 3—figure supplement 3
Characterization of CFP FLARE-D1ER.
(a) Schematic of the ER calcium sensor. (b) REF52 cells expressing CFP FLARE D1ER were permeabilized using 5 μM ionomycin and equilibrated with 3 mM EGTA to remove Ca2+ or the indicated Ca2+ …
Figure 3—figure supplement 4
Venus-cp172Venus FLARE ICUE single cell traces.
(a) Individual (gray) and average (red) cell traces for Venus-cp172Venus FLARE-ICUE (N = 40). Dashed lines above and below the average curve reflect the standard error of the mean.
Figure 4 with 5 supplements
Multiparameter Imaging with FLAREs.
(a) Time-course of a representative HEK293T cell co-expressing mCherry-mCherry FLARE-AKAR, mVenus-cp172Venus FLARE-EKAR-EV, and mCerulean3-mCerulean3 FLARE-Cameleon, with 50 μM forskolin and 100 μM …
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Figure 4—source data 1
Multiparameter imaging of FLAREs.
(a) Sheet 1, Figure 4a. Time course for multiplexed imaging of mCherry-mCherry FLARE AKAR, Venus-cp172Venus FLARE EKAR, and mCer3-mCer3 FLARE Cameleon, expressed in HEK293T cells and treated with forskolin and IBMX, EGF, and thapsigargin at t = 0 min, t = 7.5 min, and t = 32.5 min, respectively. (b) Sheet 2, Figure 4b. Time course for Venus-cp172Venus FLARE ICUE and mCer3-mCer3 FLARE Cameleon in Min6 cells, treated with TEA at t = 0 min. (c) Sheet 3, Figure 4c. Time course for Venus-cp172Venus FLARE Cameleon co-expressed with mCherry tagged hChR2-ER or mCherry alone. (d) Sheet 4, Figure 4e. Summary data of 2-photon in vivo imaging of Venus-cp172Venus FLARE Cameleon and mCherry-mCherry FLARE AKAR, in the muscle cells in the feet of live mice.
- https://cdn.elifesciences.org/articles/35458/elife-35458-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Average and single cell traces for multiplexed imaging of PKA activity, Erk activity, and calcium in HEK293-T cells.
(a) Average cell traces for mCherry-mCherry FLARE-AKAR, Venus-cp172Venus FLARE-EKAR, and mCer3-mCer3 FLARE Cameleon, expressed in HEK293T cells, and stimulated with 50 μM forskolin and 100 μM IBMX …
Figure 4—figure supplement 2
Multiplexed monitoring of PKA activity, Erk activity, and calcium in HeLa cells, with histamine stimulation.
(a) Average cell traces for mCherry-mCherry FLARE-AKAR, Venus-cp172Venus FLARE-EKAR, and mCer3-mCer3 FLARE Cameleon, expressed in HeLa cells, and stimulated with 50 μM forskolin and 100 μM IBMX at t …
Figure 4—figure supplement 3
Simultaneous monitoring of PKA and calcium in live cells with FLAREs using isoproterenol stimulation.
(a) Average cell traces for mCerulean3-mCerulean3 FLARE-AKAR, and mVenus-cp172Venus FLARE Cameleon, expressed in HEK293T cells, and stimulated with 100 nM isoproterenol at t = 0 min and 1 μM …
Figure 4—figure supplement 4
Monitoring calcium and cAMP oscillations in pancreatic β cells.
Additional cell traces for mCer3-mCer3 FLARE-Cameleon and Venus-cp172Venus ICUE, coexpressed in MIN6 cells, showing out of phase oscillations upon stimulation with TEA (N = 19).
Figure 4—figure supplement 5
Individual (gray) and average (red) cell traces for Venus-cp172Venus FLARE AKAR co-expressed with hChR2-ER, with intermittent exposures to 455 nm light (N = 11).
Dashed lines reflect the standard error of the mean.
Author response image 1
Single cell traces of EKAR2.3-EV, in HEK cells.EKAR2.3-EV with a nuclear exclusion sequence was expressed in HEK cells, and cells were stimulated with 100ng/mL epidermal growth factor (EGF) at t=0.
Author response image 2
a). Agarose beads were separately labeled with recombinant mCerulean3 or mVenus. Imaging was performed using collection conditions specific for mCerulean3 or mVenus. b). The fluorescence intensity …
Tables
Table 1
Signal to noise ratios for FLARE biosensors.
| Sensor | Signal to noise ratio (± SEM) |
|---|---|
| Venus-cp172Venus FLARE AKAR | 32 ± 2.0 (N = 32) |
| mVenus-mVenus FLARE AKAR | 10 ± 1.6 (N = 32) |
| EGFP-EGFP FLARE AKAR | 6 ± 0.6 (N = 32) |
| mCherry-mCherry FLARE AKAR | 14 ± 1.5 (N = 22) |
| mCerulean3-mCerulean3 FLARE AKAR | 3 ± 0.5 (N = 10) |
| mCerulean3-cp173 Cerulean3 FLARE AKAR | 5 ± 0.3 (N = 26) |
| Venus-cp172Venus FLARE EKAR | 17 ± 3.1 (N = 8) |
| mCherry-mCherry FLARE EKAR | 4 ± 0.8 (N = 10) |
| mCerulean3-mCerulean3 FLARE EKAR | 9 ± 2.3 (N = 9) |
| Venus-cp172Venus FLARE CKAR | 27 ± 3.3 (N = 26) |
| mCherry-mCherry FLARE CKAR | 14 ± 3.0 (N = 8) |
| mCerulean3-mCerulean3 FLARE CKAR | 9 ± 1.7 (N = 6) |
| FLARE-MLCK | 8 ± 0.8 (N = 13) |
| Venus-cp172Venus FLARE Cameleon | 19 ± 4.8 (N = 10) |
| mCherry-mCherry FLARE Cameleon | 5 ± 0.8 (N = 23) |
| mCerulean3-mCerulean3 FLARE Cameleon | 9 ± 1.5 (N = 11) |
| Venus-cp172 Venus FLARE ICUE | 19 ± 1.2 (N = 40) |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| Cell Line (Homo sapiens) | HEK293-T | RRID:CVCL_0063 | |
| Cell Line (Homo sapiens) | HeLa | RRID:CVCL_0030 | |
| Cell Line (Mus musculus) | Min6 | RRID:CVCL_0431 | |
| Cell Line (Rattus norvegicus) | REF-52 | RRID:CVCL_6848 | |
| Chemical Compound, Drug | Forskolin | LC Laboratories | F9929 |
| Chemical Compound, Drug | 3-Isobutyl-1-methylxanthine (IBMX) | Sigma | I5879 |
| Chemical Compound, Drug | H-89 | LC Laboratories | H5239 |
| Chemical Compound, Drug | Epidermal Growth Factor (EGF) | Sigma | E9644 |
| Chemical Compound, Drug | U0126 | Sigma | U120 |
| Chemical Compound, Drug | Phorbol 12-Myristate 13-Acetate (PMA) | LC Laboratories | P-1680 |
| Chemical Compound, Drug | Tetraethylammonium chloride (TEA) | Sigma | T2265 |
| Chemical Compound, Drug | ionomycin | EMD Millipore | 407951 |
| Chemical Compound, Drug | thapsigargin | Cayman Chemical | 10522 |
| Chemical Compound, Drug | histamine | Sigma | H7250 |
| Commerical Kit | Lipofectamine-2000 | Invitrogen | 11668019 |
| Commerical Kit | Polyjet | SignaGen | SL100688 |
| Recombinant DNA Reagent | pCDNA3 AKAR4 | PMID: 20838685 | |
| Recombinant DNA Reagent | pCDNA3 EKAR-EV | PMID: 21976697 | |
| Recombinant DNA Reagent | pCDNA3 CKAR1 | PMID: 12782683 | |
| Recombinant DNA Reagent | FRET MLCK sensor | PMID: 15071183 | |
| Recombinant DNA Reagent | pCDNA3 YC3.6 Cameleon | PMID: 10051607 | |
| Recombinant DNA Reagent | pCDNA3 ICUE3 | PMID: 19603118 | |
| Strain (Escherichia coli) | BL-21 Codon Optimized Plus | New England Biolabs | C2527 |
| Software | FIJI | RRID:SCR_014294 | |
| Software | MetaFluor | RRID:SCR_002285 | |
| Software | Micromanager | RRID:SCR_000415 | |
| Software | Zeiss Actiovision | RRID:SCR_002677 | |
| Software | MATLAB | RRID:SCR_001622 | |
| Software | GraphPad Prism | RRID:SCR_002798 |
