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Single-molecule FRET imaging of GPCR dimers in living cells

Abstract

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

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Fig. 1: Imaging and tracking receptor dimers in living mammalian cells by smFRET.
Fig. 2: Agonist-induced conformational dynamics in Sf-mGluR2 dimers.
Fig. 3: Comparing the dimerization of select TM proteins by TIRF-based smFRET imaging and confocal-based PIE-FCCS.
Fig. 4: Summary of the smFRET-RAP method and representative Sf-mGluR2 data.
Fig. 5: Summary of smFRET-RAP data for Sf-SecR and Sf-MOR.

Data availability

The raw image data generated and analyzed that support the findings of this study are available from the corresponding author upon reasonable request. These image data are not deposited in a public database because of their large file sizes. Source data are provided with this paper.

Code availability

The smCellFRET data analysis pipeline is freely available for academic use. The software and updated versions can be downloaded at http://innovation.columbia.edu/technologies/CU15268. Other software used to collect and analyze data for this work as described in the Methods either was published previously or is commercially available.

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Acknowledgements

This work was supported by NIH grant MH54137 (J.A.J.), the Hope for Depression Research Foundation (J.A.J.), the Lieber Center for Schizophrenia Research (J.A.J.), the Brain and Behavior Research Foundation NARSAD Young Investigator Award (W.B.A.), NIH grant R15EY024451 (A.W.S.), the National Science Foundation under grant number CHE-1753060 (A.W.S.), NIH grant R35GM119619 (K.J.), NIH grant 7R01GM098859-09 (S.C.B.) and the UTSW Endowed Scholars Program (K.J.). This work was supported, in part, by the Single-Molecule Imaging Center at St. Jude Children’s Research Hospital. We thank M. Dawoud for technical assistance, B. Williams for analysis of online RNA-seq data in CHO cells, S. Mondal and H. Weinstein for discussion related to the measurement of diffusion, A. Vega for discussion related to DC-MSS, G. Schütz, M. Brameshuber and C. Bodner for discussion related to single-molecule imaging at high surface expression conditions, I. Correa for discussion related to SNAPf labeling and E. Stevens for assistance with figure illustrations. This work is dedicated to the memory of Y. Zhao.

Author information

Affiliations

Authors

Contributions

W.B.A., S.C.B. and J.A.J. wrote the manuscript, with contributions from all of the authors. W.B.A., P.G. and J.A.J. designed single-molecule TIRF and smFRET imaging experiments. W.B.A., M.D.H. and K.G.H. generated the stable CHO cell lines. W.B.A. and M.D.H. performed all live-cell single-molecule imaging experiments, and P.G., W.B.A., J.M., M.D.H., D.S.T. and S.M. analyzed the data with input from S.C.B. and J.A.J. A.W.S., G.T.G. and M.D.M. designed the PIE-FCCS experiments. G.T.G., M.J.K. and M.D.M. performed the PIE-FCCS experiments. A.G. and W.B.A. collected and analyzed the BRET-based cell assay data. Z.Z. synthesized and characterized the self-healing fluorophores. A.K.P. prepared the recombinantly expressed and purified SNAPf–fluorophore conjugates and collected the photophysical data for these samples. P.G. developed the SMCellFRET pipeline for tracking smFRET and further analysis with input from W.B.A., J.M., K.J., S.M., D.S.T., S.C.B. and J.A.J. D.S.T. performed the fluorescence cross-correlation analysis. J.A.J. supervised the project.

Corresponding authors

Correspondence to Scott C. Blanchard or Jonathan A. Javitch.

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Competing interests

S.C.B. has an equity interest in Lumidyne Technologies. The other authors have no competing interests.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Nature Methods thanks Emanuel Margeat, Michael Börsch, and the other, anonymous reviewer for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Expression and labeling of functional Sf-mGluR2 with self-healing fluorophores in living CHO cells.

a, The SNAPf tag is a modified O6-alkylguanine-DNA alkyltransferase enzyme that forms covalent linkages with benzylguanine (BG)-fluorophores. b, Dose-response curve for bioluminescence resonance energy transfer (BRET)-based cAMP inhibition assay confirming Sf-mGluR2 functionality. Global fits from three independent experiments each performed in triplicate. Error bars represent standard error of the mean (SEM). The mean Log EC50 with standard error is shown. c, Schematic of LEx-FITR CHO cells expressing the tet repressor (TetR) and with integrated receptor cDNA under control of the crippled CMV promoter (PcrCMV), two tetracycline operator 2 (2XTO2) sites, and weak Kozac sequence. Note that LEx-FITR cells were selected for a Flp-In site that leads to very low basal expression after receptor cDNA integration. d, Chemical structures of LD555p-BG and LD655-BG. Absorption (left plots) and emission (right plots) spectra of recombinantly expressed and purified SNAPf labeled with e, LD555p and f, LD655.

Source data

Extended Data Fig. 2 Quantification of the surface density of labeled Sf-mGluR2.

a, Representative initial image of a CHO cell containing donor and acceptor labeled Sf-mGluR2 of 16 cells generated by 532-nm and 640-nm dual excitation taken prior to smFRET imaging. Single-particle detection (purple circles) was used to quantify the number of particles within a region of interest (yellow line). Scale bar, 5 µm. b, Surface densities prior to smFRET imaging of cells labeled with donor and acceptor (Don/Acc) as well as with acceptor only (Acc-only) or with donor only (Don-only). Dots represent the number of acceptors or donors per area for each cell. Box plots indicate the median (central line) and interquartile range (IQR) (lower and upper box lines represent the 25- and 75-percentiles, respectively) while the whiskers represent those points that fall within 1.5 x IQR. The median density of total (acceptor + donor) labeled receptors was 0.30 receptors/μm2 (donor-to-acceptor ratio ~1:1). As expected, donor- and acceptor-only samples show labeling with only the fluorophore indicated.

Source data

Extended Data Fig. 3 Fluorescence and smFRET data for donor and acceptor labeled Sf-mGluR2 immobilized under fixed cell conditions.

a, Representative fluorescence (top)- and FRET (bottom) time traces for individual receptors. b, Distributions of the total intensity (donor + acceptor) and acceptor intensity during smFRET. The distributions were fit to a single gaussian function, yielding a mean total intensity of 458 photons/frame (full width at half maximum (FWHM) of 218) and a mean acceptor intensity of 279 photons/frame (FWHM of 288). c, Lifetime of smFRET events for Sf-mGluR2 in fixed cells. The lifetime distribution was fit to a single exponential to produce the decay constant τ. The data in panels b and c are derived from 124 molecules and a total of 16 cells.

Source data

Extended Data Fig. 4 Characterizing fluorescence and smFRET data for Sf-mGluR2 in the plasma membrane of living cells.

a, Total fraction of time spent in (left) and diffusion coefficients (right) for immobile (imm), confined (conf), free, and directed (dir) diffusion states assigned by DC-MSS. Dots represent individual cell means and the middle and upper/lower lines depict the overall mean (values shown) and standard deviation, respectively, for 16 cells. b, Distributions of the total (donor + acceptor) and acceptor intensities during smFRET. Histograms comprised of 5,546 freely diffusing smFRET trajectories from 16 cells were fit with a single-state gaussian model, yielding mean total and acceptor intensities of 457 (FWHM of 284) and 190 (FWHM of 135) photons/frame, respectively. c, Distribution of freely diffusing smFRET events per cell for receptor labeled with donor and acceptor (Don/Acc) (16 cells) compared to those with acceptor-only (16 cells) and donor-only (22 cells). Dots represent the total number of freely diffusing smFRET trajectories (including freely diffusing segments from smFRET trajectories with more than one diffusion state) per area for each cell. Box plot details are described in the legend of Extended Data Fig. 2b. One-way ANOVA (DF = 53; F-value = 92.5) and Tukey post-hoc comparison were performed to obtain p-values (****p « 0.0001; not significant (n.s.) = 0.997). The sum of the mean number of events per cell for the controls represent ~1% of those from Don/Acc Sf-mGluR2. d, Representative smFRET trajectories and fluorescence- and FRET-time traces for Sf-mGluR2 in the absence of ligand (apo state) without and e, with anticorrelation. Here and elsewhere, smFRET trajectories are shown to the left of their fluorescence (red and green traces indicating the intensities are derived from acceptor and donor tracks) and FRET traces. f, FRET-efficiency histograms fit with a single-(top) or two (bottom)-state gaussian model from traces without (top) and with anticorrelation (bottom) containing donor and acceptor labeled-mGluR2. The histograms are comprised of the number of trajectories (n) indicated from 6 cells. Each bar height represents the mean count of FRET values calculated from 6 cell samples. The length of the error bars corresponds to 1 s.d. from the mean. g, Distribution of the duration of smFRET events of 4,800 freely diffusing smFRET trajectories from 16 cells with single-exponential decay constant τ. h, Pearson correlation coefficients between donor and acceptor fluorescence traces were calculated for each segment and are shown as a histogram for the immobile (black), confined (magenta), and freely diffusing (blue) motion types. Lines are spline interpolations to facilitate comparison between conditions. Values in the legend correspond to the ensemble average correlation values. (i) FRET efficiency histogram comprised of immobile/confined segments for Sf-mGluR2 labeled with donor and acceptor. The histogram is fit with a two-state Gaussian model and consists of 93 immobile/confined segments from trajectories that also showed free diffusion obtained from 6 different cells. Error bars are described in the legend of Extended Data Fig. 4f.

Source data

Extended Data Fig. 5 SmFRET data for Sf-mGluR2 dimers diffusing within the plasma membrane of living cells.

Representative smFRET trajectories and their corresponding fluorescence- and FRET-time traces for individual receptors in the presence of a, 15 µM and b, 100 µM glutamate (Glu) as well as c, those showing transitions to the 0.84 FRET state (top, apo condition; bottom, 15 µM Glu condition). d, Pearson correlation coefficients between donor and acceptor fluorescence traces were compiled into histograms for trajectories obtained in the absence of ligands (blue), or in the presence of 15 µM Glu (green) or 100 µM Glu (red). Lines are spline interpolations to facilitate comparison between conditions. Values in the legend correspond to the ensemble average correlation values.

Source data

Extended Data Fig. 6 Characterization of functional Sf-MOR, Sf-SecR, and Sf-mGluR2 compared to controls in living cells.

a, Dose-response curves for BRET-based cAMP inhibition and generation assays confirming Sf-MOR (top) and Sf-SecR (bottom) functionality, respectively. Curve fitting details are described in Extended Data Fig. 1b legend. b, Surface densities prior to smFRET imaging of donor and acceptor labeled samples for smFRET studies. Dots represent the number of acceptor (nAcc) or donor (nDon) particles per area for single cells. Box plot details are described in the legend of Extended Data Fig. 2b. The densities for Sf-mGluR2 are reproduced from Extended Data Fig. 2b for comparison. The median density of total labeled (acceptor + donor) TM proteins ranged from 0.28 – 0.36 molecules/μm2. c, Distribution of smFRET events per cell for Sf-mGluR2 labeled with donor and acceptor (Don/Acc) (16 cells) compared to those for acceptor-only (16 cells) and donor-only (22 cells) controls as determined by the NLT analysis criteria. Dots represent the number of smFRET trajectories per area for each cell. Box plot details are described in the legend of Extended Data Fig. 2b. One-way ANOVA (DF = 53; F-value = 75.5) and Tukey post-hoc comparison were performed to obtain p-values (****p « 0.0001; n.s. = 0.996). The sum of the mean number of events per cell for the controls represent < 2% of those from Don/Acc Sf-mGluR2. d, Distribution of the duration of smFRET events of 2,695 smFRET trajectories for Sf-mGluR2 from 16 cells with the single-exponential with decay τ.

Source data

Extended Data Fig. 7 Overview of the PIE-FCCS method.

a, Schematic of the PIE-FCCS setup. Blue and green excitation beams, split from the same source, travel along fibers of different lengths to interleave the pulse arrival times. The diffraction-limited beams are focused at the cell surface and photons emitted from fluorescently labeled TM proteins diffusing through the laser focus are collected by the objective and directed to single photon detectors coupled to a TCSPC device. b, Pulsed interleaved excitation allows for separate time gating of green and red fluorophore emission readings that are time-tagged by the TCSPC device. c, A representative Sf-mGluR2 expressing CHO cells labeled with ATTO488-BG (left) and DY549P1-BG (right) in a ~1:1 ratio. White squares indicate approximate position and size of the laser focus during PIE-FCCS data collection. Scale bars, 10 µm. d, Example PIE-FCCS data from a single cell for Sf-mGluR2. Green and red dots are the autocorrelation functions (ACFs) obtained from fluorescence fluctuations in the green and red detection channels, respectively, while blue dots are the cross-correlation function (CCF) from the green and red co-diffusing species. The solid lines are model fits used to calculate fraction correlated (fc) as described in the Methods. e, Total surface densities of labeled samples for PIE-FCCS studies. Dots represent the total number of labeled molecules per area for single cells. Box plot details are described in the legend of Extended Data Fig. 2b.

Source data

Extended Data Fig. 8 Representative PIE-FCCS data curves for each construct.

PIE-FCCS data as described in Extended Data Fig. 7d from three representative cells for a, Sf-TM-LDL, b, Sf-mGluR2, c, Sf-Δ2Δ, d, Sf-MOR, and e, Sf-SecR.

Source data

Extended Data Fig. 9 SmFRET-RAP data for Sf-mGluR2.

a, Relationship between the number of acceptor and donor particles (nParticles) recovered 2 – 3 minutes after photobleaching and the total-background corrected acceptor and donor fluorescence per cell area. The number of cells for each point is 8. b, Surface densities of donor and acceptor labeled receptors before bleaching for the smFRET-RAP experiments (the median density of total labeled (acceptor + donor) receptors was ~ 4.0 molecules/μm2 (donor-to-acceptor ratio ~1:1)) compared to (c) those used for smFRET at lower expression levels reproduced from Extended Data Fig. 2b for comparison. Dots represent the number of acceptors (nAcc) and donors (nDon) per area for individual cells. Box plot details are described in the legend of Extended Data Fig. 2b. d, Representative acceptor and donor image sequence during smFRET with corresponding smFRET trajectory (red and green lines). Scale bar, 5 µm. Purple arrow at 10.64 s indicates a second donor particle that overlaps briefly with the particle showing smFRET. These occurrences do not influence the number of FRET events or their lifetime. e, Representative smFRET trajectory and fluorescence- and FRET-time traces derived from the image sequence in (d) where the donor and acceptor emission are anticorrelated upon acceptor photobleaching. f, Distribution of the duration of smFRET-RAP events comprised of 7,529 smFRET-RAP trajectories from 8 cells with single-exponential decay constant τ.

Source data

Extended Data Fig. 10 SmFRET-RAP data for Sf-SecR and Sf-MOR.

a, Acceptor (top) and donor (bottom) labeled receptor densities before photobleaching for smFRET-RAP (left panel) compared to those used for smFRET at lower receptor expression levels (right panel) reproduced from Extended Data Fig. 6b. Dots represent the number of acceptor (nAcc) or donor (nDon) particles per area for single cells. Box plot details are described in the legend of Extended Data Fig. 2b. b, c, TIRF images of representative CHO cells expressing labeled b, Sf-SecR from 7 cells and (c) Sf-MOR from 7 cells before donor and acceptor photobleaching (left panel), ~30 seconds after photobleaching (middle panel), and ~2 – 3 minutes after photobleaching (right panel) showing the recovery of labeled receptors (scale bar, 5 μm). d, Representative trajectories and sensitized acceptor intensity time traces for Sf-SecR. The top trajectory and trace are derived from the image sequence shown in Fig. 5b. e, Duration of smFRET events of SecR interactions determined from the tracking duration of sensitized acceptor trajectories. The distribution comprised of 4,232 trajectories from 21 cells was fit to a single exponential with decay constant (τ).

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Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, Tables 1–7 and Notes 1–8

Reporting Summary

Supplementary Video 1

Representative movie of a live CHO cell containing acceptor- and donor-labeled Sf-mGluR2 imaged by smFRET. The first 500 frames acquired for the cell shown in Fig. 1b show acceptor particles during FRET in the left channel and directly excited donors in the right channel. Scale bar, 5 µm.

Supplementary Video 2

Movie of an enlarged region (the same enlarged region shown in Fig. 1b) of the cell in Supplementary Video 1. Acceptor particles during FRET and directly excited donors are shown in the left and right channels, respectively. Scale bar, 5 µm.

Supplementary Video 3

Representative movie of image sequences with overlaid trajectories for individual acceptor- and donor-labeled Sf-mGluR2 proteins diffusing at the cell surface imaged by smFRET, shown in Fig. 1c. The movies show the acceptor during FRET (left channel) and its corresponding donor (right channel). Tracking for the acceptor and donor is shown as red and green lines during the movie.

Supplementary Video 4

Representative movie of image sequences with overlaid trajectories for individual acceptor- and donor-labeled Sf-mGluR2 proteins diffusing at the cell surface imaged by smFRET. The movies show the acceptor during FRET (left channel) and its corresponding donor (right channel). Tracking for the acceptor and donor is shown as red and green lines during the movie.

Supplementary Video 5

Representative movie of image sequences with overlaid trajectories for individual acceptor- and donor-labeled Sf-mGluR2 proteins diffusing at the cell surface imaged by smFRET. The movies show the acceptor during FRET (left channel) and its corresponding donor (right channel). Tracking for the acceptor and donor is shown as red and green lines during the movie.

Supplementary Video 6

Representative movie of the acceptor during FRET for Sf-SecR imaged by smFRET-RAP in live CHO cells. The acceptor trajectory is shown in red.

Supplementary Video 7

Representative movie of the acceptor during FRET for Sf-SecR imaged by smFRET-RAP in live CHO cells. The acceptor trajectory is shown in red.

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Asher, W.B., Geggier, P., Holsey, M.D. et al. Single-molecule FRET imaging of GPCR dimers in living cells. Nat Methods 18, 397–405 (2021). https://doi.org/10.1038/s41592-021-01081-y

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