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

Published online:


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.

  • Subscribe to Nature Biotechnology for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


Protein Data Bank


  1. 1.

    et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

  2. 2.

    et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

  3. 3.

    et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

  4. 4.

    , & A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

  5. 5.

    , , & In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

  6. 6.

    et al. Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators. Science 347, 755–760 (2015).

  7. 7.

    et al. Rationally improving LOV domain-based photoswitches. Nat. Methods 7, 623–626 (2010).

  8. 8.

    et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

  9. 9.

    et al. The genetic design of signaling cascades to record receptor activation. Proc. Natl. Acad. Sci. USA 105, 64–69 (2008).

  10. 10.

    et al. Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148, 583–595 (2012).

  11. 11.

    , , & The P1′ specificity of tobacco etch virus protease. Biochem. Biophys. Res. Commun. 294, 949–955 (2002).

  12. 12.

    et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993–1000 (2001).

  13. 13.

    et al. Labelling and optical erasure of synaptic memory traces in the motor cortex. Nature 525, 333–338 (2015).

  14. 14.

    , & LOV-based optogenetic devices: light-driven modules to impart photoregulated control of cellular signaling. Front. Mol. Biosci. 2, 18 (2015).

  15. 15.

    , , & Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009).

  16. 16.

    , , , & Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat. Struct. Biol. 7, 215–219 (2000).

  17. 17.

    et al. Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer. Proc. Natl. Acad. Sci. USA 105, 542–547 (2008).

  18. 18.

    et al. Unfolding of the C-terminal Jα Helix in the LOV2 photoreceptor domain observed by time-resolved vibrational spectroscopy. J. Phys. Chem. Lett. 7, 3472–3476 (2016).

  19. 19.

    & Factors that control the chemistry of the LOV domain photocycle. PLoS One 9, e87074 (2014).

  20. 20.

    et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).

  21. 21.

    et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

  22. 22.

    et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

  23. 23.

    et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).

  24. 24.

    et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

  25. 25.

    , , , & Transgenic inhibition of synaptic transmission reveals role of CA3 output in hippocampal learning. Science 319, 1260–1264 (2008).

  26. 26.

    , , , & Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl. Acad. Sci. USA 104, 5163–5168 (2007).

  27. 27.

    et al. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 3, 985–993 (2006).

  28. 28.

    et al. Temporally precise labeling and control of neuromodulatory circuits in the mammalian brain. Nat. Methods 14, 495–503 (2017).

  29. 29.

    et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–385 (2012).

  30. 30.

    , , , & Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron 78, 773–784 (2013).

  31. 31.

    , , & Localization of a stable neural correlate of associative memory. Science 317, 1230–1233 (2007).

  32. 32.

    et al. A robust activity marking system for exploring active neuronal ensembles. eLife 5, 2 (2016).

  33. 33.

    et al. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).

  34. 34.

    et al. Ca2+ Indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 13, 521–530 (2006).

  35. 35.

    & N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry 46, 14001–14009 (2007).

  36. 36.

    et al. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755–768 (2006).

  37. 37.

    , , , & Investigating models of protein function and allostery with a widespread mutational analysis of a light-activated protein. Biophys. J. 105, 1027–1036 (2013).

  38. 38.

    , & The amino-terminal helix modulates light-activated conformational changes in AsLOV2. J. Mol. Biol. 419, 61–74 (2012).

  39. 39.

    et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

  40. 40.

    et al. Proteomic analysis of unbounded cellular compartments: synaptic clefts. Cell 166, 1295–1307.e21 (2016).

Download references


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.

Author information


  1. Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California, USA.

    • Wenjing Wang
    • , Mateo I Sanchez
    •  & Alice Y Ting
  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Wenjing Wang
    • , Tanyaporn Pattarabanjird
    • , Mateo I Sanchez
    •  & Alice Y Ting
  3. Picower Institute for Learning and Memory and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Craig P Wildes
    • , Gordon F Glober
    • , Gillian A Matthews
    •  & Kay M Tye


  1. Search for Wenjing Wang in:

  2. Search for Craig P Wildes in:

  3. Search for Tanyaporn Pattarabanjird in:

  4. Search for Mateo I Sanchez in:

  5. Search for Gordon F Glober in:

  6. Search for Gillian A Matthews in:

  7. Search for Kay M Tye in:

  8. Search for Alice Y Ting in:


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.

Competing interests

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

Corresponding authors

Correspondence to Kay M Tye or Alice Y Ting.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary figures 1–13 and Supplementary tables 1–2.