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A light- and calcium-gated transcription factor for imaging and manipulating activated neurons


Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

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Figure 1: Engineering the calcium and light responses of FLARE.
Figure 2: Directed evolution of LOV domain to improve light gating in FLARE.
Figure 3: FLARE optimization and testing in neurons.
Figure 4: Functional reactivation of neurons marked by FLARE and in vivo testing.

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We thank J. Einstein and A. Draycott for neuron cultures. J. Einstein also assisted with cloning and some imaging assays. F. Zhang, S. Konermann, and M. Brigham provided AAV vectors, AAV protocols, and guided us on the setup of our light stimulation device. G. Liu built the LED light box. M. Heidenreich advised us on preparation of concentrated AAVs. T.J. Wardill and L. Looger provided electrode information. H. Wang and M. Djuristic assisted with electrical stimulation setup. P. Han assisted with statistical analysis of the in vivo data. FACS experiments were performed at the Koch Institute Flow Cytometry Core (MIT). The TEVp gene was a gift from the Waugh laboratory (National Cancer Institute). GCaMP5f was a gift from L. Looger, Janelia Research Campus. A.Y.T. received funding from MIT and Stanford. K.M.T. is a New York Stem Cell Foundation Robertson Investigator and McKnight Scholar and this work was supported by funding from the JPB Foundation, the PIIF and PIIF Engineering Award, R01-MH102441-01 (NIMH), and NIH Director's New Innovator Award DP2-DK-102256-01 (NIDDK). G.A.M. was supported by a fellowship from the Charles A. King Trust Postdoctoral Research Fellowship Program, Bank of America, N.A., Co-Trustees.

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Authors and Affiliations



W.W. performed all experiments except those noted. W.W. and T.P. together performed the LOV directed evolution. M.I.S. generated AAV viruses and measured eLOV recovery kinetics. C.P.W. and K.M.T. designed the in vivo experiments. C.P.W., G.F.G., and G.A.M. performed the in vivo experiments. W.W. and M.I.S. analyzed the in vivo data. W.W. and A.Y.T. designed the research, analyzed the data, and wrote the paper. All authors edited the paper.

Corresponding authors

Correspondence to Kay M Tye or Alice Y Ting.

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Competing interests

A.Y.T. and W.W. have filed a patent application covering some aspects of this work.

Integrated supplementary information

Supplementary Figure 1 Summary of published TEV protease catalytic constants.

The S219V mutation in TEV protease prevents autolysis at position 218. FLARE uses truncated TEV protease (Δ220-242) and the TEV cleavage site (TEVcs) with X=M. Tango uses wild type TEV protease with TEVcs X=L3.

Supplementary Figure 2 Library progression during directed evolution of the LOV domain.

Related to Fig. 2. Re-amplified yeast cultures following each round of selection were assayed under matched conditions. The original AsLOV2 (G528A/N538E mutant4) and final eLOV are also shown for comparison. To evaluate dark state leak, yeast were treated with ~30 μM wild-type TEV protease in the dark for 3 hours, then stained with anti-Flag and anti-HA antibodies as in Fig. 2C. To evaluate TEVcs accessibility in the light state, yeast were treated with ~30 μM TEV protease under a broad wavelength light source for 1 hour, then stained with antibodies. The red polygons show the FACS sorting gates used in negative selections; green polygons depict positive selection FACS gates. This experiment has been performed once.

Supplementary Figure 3 Sequencing analysis of yeast clones from LOV directed evolution experiment.

12 clones were sequenced from the original LOV library, and 15 clones from the final round of selection (round 6). Mutations with respect to the original LOV2 protein4 are indicated. Some clones were the original LOV2 (first column), some contained silent mutations, and one had a mutation outside the LOV2 gene. This experiment has been performed once.

Supplementary Figure 4 FACS analysis of specific LOV mutants on yeast.

(A) Analysis of five LOV mutants enriched after 6 rounds of selection. Original LOV2 is shown for comparison. Each clone is evaluated for dark state protection and light state cleavage as in Fig. 2C and Supplementary Fig. 2. Numbers in top right of each graph give the percentage of cells in Q2 quadrant (out of total cells in Q2 + Q4). (B) Five designed LOV mutants based on manual combination of mutations in (A). Clones were evaluated on yeast as in (A). (C) LOV2 structure (PDB:2V1A5) highlighting proximity between H117 in the LOV core and E123 in the Jα helix. eLOV has an H117R mutation, which may interact with E123 to help stabilize eLOV in the dark state, leading to improved caging. (A) and (B) have each been replicated once.

Supplementary Figure 5 Comparison of original LOV and eLOV in HEK 293T cells.

Same as Fig. 2D, but with additional fields of view, and immunofluorescence staining of the transcription factor component (anti-HA) as well. (A) is original AsLOV2 and (B) is eLOV. DIC, Differential Interference Contrast image. Scale bars, 20 μm. (A) and (B) have each been replicated 2 times.

Supplementary Figure 6 Screening alternative TEVp cleavage site (TEVcs) sequences in HEK cells.

(A) Summary of results. In Fig. 1, we compared X=L (from Tango, low affinity) and X=Y (high affinity) in TEVcs. Here, we expanded our testing of TEVcs sequences to X = Y, A, N, H, M, Q and W. TEVcs sequences were inserted into the FLARE construct TM(CD4)-CaMbp(M2)-eLOV-TEVcs-Gal4. This was introduced by PEI max transfection into HEK cells along with CaM-TEVp(truncated) and UAS-Citrine. High calcium (5 minutes) and light conditions were the same as in Fig. 1F. Signal ratios were based on mean Citrine intensities across >2000 cells from 10 fields of view per condition. Errors, s.e.m. (B) Fluorescence images for the X=M and X=Y constructs in (A). Citrine channels are shown at 10x magnification, 5 fields of view per condition. Scale bars, 100 μm. This experiment has been replicated once. P1′=M gave improvements in both high/low calcium signal ratio and light/dark signal ratio, mainly due to higher Citrine signal in the +light +high Ca2+ state. This is consistent with previous literature showing that P1′=M gives 6-fold and 13-fold faster kcat, respectively, for cleavage by TEVp compared to P1′=Y and P1′=L2 (Supplementary Fig. 1).

Supplementary Figure 7 Sequences of final FLARE components.

The FLARE TF component (top), protease component (middle), and FLARE-regulated transgene (bottom) were packaged into AAVs for use in neuron culture (Fig. 3-4) and in vivo (Fig. 4).

Supplementary Figure 8 Media change generates calcium rises in cultured neurons.

DIV19 cortical rat neurons expressing the calcium indicator GCaMP5f10 were imaged before and after replacement of half of the media volume with fresh media of identical composition (neurobasal supplemented with B27, Glutamax and penicillin/streptomycin). Time courses show that media change leads to sustained elevation of cytosolic calcium. Scale bars, 100 μm. This experiment has been replicated two times.

Supplementary Figure 9 FLARE sensitivity/time course.

Same as Fig. 3E, but with additional time points and accompanying fluorescence images. (A) Summary graph of FLARE response as a function of stimulation time. 90% of the culture media was replaced one time (at t=0), and then blue light (473 nm LED, 60 mW/cm2, 10% duty cycle (0.5 sec light every 5 sec)) was applied for 2-30 minutes, as indicated. Error bars, s.e.m. (B) Fluorescence images for datapoints in (A). For each condition, 5 fields of view are shown. Scale bars, 100 μm. This experiment has been replicated once.

Supplementary Figure 10 Characterization of purified LOV proteins.

(A) SDS-PAGE (9%) gel electrophoresis of purified LOV proteins. WT-LOV2 (ref. 11) and original LOV2 (G528A/N538E mutant4) are both from Avena sativa. eLOV is evolved from original LOV2 (Fig. 2). (B) UV-Vis absorbance spectra of LOV variants before (black line) and after (blue line) 1 minute of irradiation with 470 nm light. (C) Absorbance timecourses to measure the kinetics of LOV re-set to the dark state after 1 minute of blue light exposure. Time constants calculated from single exponential fits. This experiment has been replicated once.

Supplementary Figure 11 Light- and activity-dependent expression of ChrimsonR-mCherry in vivo, via FLARE.

(A) This graph is an alternative presentation of the results of the in vivo experiment in Fig. 4e. Each point represents mCherry sum intensity across a single brain section (from one dark or light-exposed hemisphere). Seven consecutive sections were collected and analyzed per animal. Images (mCherry + BFP channels) of all sections plotted in the first two columns are shown in Supplementary Fig.12. (B) Control experiment showing that implanted optical fiber does not affect FLARE. Analysis of mCherry expression in three FLARE-expressing animals prepared as in Fig. 4c. All animals were subjected to wheel running, and light was delivered to the left hemisphere but not the right hemisphere. Animal 1 had fibers implanted in both sides of the brain. Seven consecutive brain sections were imaged and quantified per animal. Light dependence was significant in all three animals (Kolmogorov-Smirnov Test, one-sided, N=7, p < 0.01; p < 0.001). This experiment has been performed once.

Supplementary Figure 12 Confocal fluorescence images showing FLARE activation in the mouse brain.

ChrimsonR-mCherry expression and corresponding BFP (FLARE expression marker) images in brain sections from mice treated as in Fig. 4c. Each row shows 7 consecutive brain sections from one animal. Scale bars, 200 μm.

Supplementary Figure 13 FLARE drives activity- and light-dependent expression of APEX2 peroxidase.

Cultured rat cortical neurons were infected at DIV13 with AAV viruses encoding FLARE components: TRE-Citrine-APEX2; TM(NRX)-CaMbp(M2)-eLOV-TEVcs-tTA-VP16; and CaM-TEVp(truncated). At DIV18, neurons were incubated in the dark or exposed to blue light for 10 minutes (467 nm, 10% duty cycle, 60 mW/cm2). Activity stimulation was via 90% media change. 18 hours later, neurons were fixed and stained with diaminobenzidine (DAB) and H2O2 to visualize APEX2 activity. APEX2 catalyzes the polymerization and local deposition of DAB, which in turn recruits electron-dense osmium to enable electron microscopy of APEX2-expressing cells12,13. Scale bar, 100 μm.

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Supplementary figures 1–13 and Supplementary tables 1–2. (PDF 2568 kb)

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Wang, W., Wildes, C., Pattarabanjird, T. et al. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. Nat Biotechnol 35, 864–871 (2017).

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