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A general approach for engineering RTKs optically controlled with far-red light

Abstract

Regulation of receptor tyrosine kinase (RTK) activity is necessary for studying cell signaling pathways in health and disease. We developed a generalized approach for engineering RTKs optically controlled with far-red light. We targeted the bacterial phytochrome DrBphP to the cell surface and allowed its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. Systematic optimization of these constructs has resulted in optically regulated epidermal growth factor receptor, HER2, TrkA, TrkB, FGFR1, IR1, cKIT and cMet, named eDrRTKs. eDrRTKs induced downstream signaling in mammalian cells in tens of seconds. The ability to activate eDrRTKs with far-red light enabled spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays. We validated eDrTrkB performance in mice and found that minimally invasive stimulation in the neocortex with penetrating via skull far-red light-induced neural activity, early immediate gene expression and affected sleep patterns.

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Fig. 1: Workflow of the eDrRTK design.
Fig. 2: Induction of Elk-1-dependent luciferase reporter expression by eDrRTKs.
Fig. 3: Phosphorylation of eDrRTKs and ERK1/2 on short-term action of FR light.
Fig. 4: Activation of eDrTrkB in neuronal cells.
Fig. 5: Amounts of NREM sleep increased in mice expressing eDrTrkB in the cerebral cortex following exposure to FR light.
Fig. 6: NeuN, mCherry and cFos expression in the somatosensory cortex of mice injected with AAV9 encoding mCherry-eDrTrkB-CW3SL.

Data availability

The main data supporting the findings of this study are available within the article and its supplementary information. The main resultant plasmids encoding eDrRTKs are deposited in the Addgene depository (nos. 183994–184001). The code used in this study for LED switching is available at https://docs.arduino.cc/built-in-examples/basics/Blink. Source data are provided with this paper.

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Acknowledgements

We thank J. Ihalainen (University of Jyväskylä, Finland) for the DrBphP gene and D. Lindholm (University of Helsinki, Finland) for the cell lines. This work was supported by the grants GM122567, AG061774 and NS106406 from the US National Institutes of Health and 322226 from the Academy of Finland.

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Contributions

A.V.L. engineered and characterized the optogenetic constructs. S.T. and C.Y. performed animal and histochemical studies. V.V.V. planned and directed the project and together with A.V.L. and D.G. designed the experiments, analyzed the data and wrote the manuscript. All authors revised the manuscript.

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Correspondence to Vladislav V. Verkhusha.

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Nature Methods thanks Bianxiao Cui, Benjamin Rost and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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.

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Extended data

Extended Data Fig. 1 Structural predeterminants of opto-RTK engineering.

(a) Various hypotheses of RTK activation. Top: RTK dimerization: inactive RTKs exist as monomers and ligand binding causes their dimerization and consequent activation. Bottom: Rotational coupling: inactive RTKs exist as preformed dimers and ligand binding causes conformational changes resulting in RTK activation. (b) Structural changes occurring in EGFR receptor. Left: EGFR receptor exists as a preformed inactive dimer. Right: EGF ligand binding causes conformational changes and EGFR activation. Ab initio models of EGFR in active and inactive states are modified with permission from15. (c) Light-induced conformational changes in DrBphP–PCM obligate dimer cause distance increase between C-termini of DrBphP-PCM protomers17. (d) Schematic representation of proposed opto-RTK design (top) and mechanism of its activation (bottom). DrBphP-PCM targeted to the extracellular surface by Igκ signaling peptide is connected to cytoplasmic RTK domain (cytoRTK) via transmembrane (tmRTK) domain. In darkness or near-infrared light, opto-RTK remains inactive. Far-red light causes DrBphP-PCM conformational changes, which are transmitted to cytoplasmic RTK domains, causing their re-orientation and trans-phosphorylation.

Extended Data Fig. 2 Engineering and characterization of opto-EGFR and opto-HER2 prototypes.

(a) Left: Ligand binding causes RTK autophosphorylation, interaction with Grb2 and SOS, and results in activation of ERK1/2 pathway consisting of RAS, RAF, MEK, and ERK1/2 kinases. Consequently, ERK1/2 activation leads to immediate early gene (IEG) expression driven by transcription factor Elk-1. Right: Likewise, activation of chimeric opto-RTK with far-red light leads to activation of ERK1/2 pathway and induction of Elk-1 dependent IEG expression. (b) Scheme of luciferase reporter assay. Top: ERK1/2 is inactive, and Elk-1 fused to Gal4DBD is monomeric and inactive. Bottom: ERK1/2 is active, and activated ERK1/2 phosphorylates Elk-1. Phosphorylated Elk-1-Gal4DBD fusion dimerizes, binds to 5x UAS sequence, and drives Firefly luciferase (FLuc) reporter expression. (c, d) Light-induced activation of Elk-1-dependent Firefly luciferase reporter expression by opto-EGFR (c) and opto-HER2 (d) prototypes in PC6-3 cells. Renilla luciferase (RLuc) was co-transfected as a normalization control. In the darkness, Firefly luciferase expression is suppressed. 660 nm light activates eDrRTKs and upregulates Firefly luciferase expression. Expression of Firefly and Renilla luciferases was analyzed after 24 h of illumination. Relative luciferase expression activity (RLA) is shown as a ratio of the Firefly luciferase expression relative bioluminescence units to the Renilla luciferase expression relative bioluminescence units (FLuc/RLuc). 25 µM BV was added to the culture medium in all experiments. Data are presented as mean values, error bars represent standard deviation (s.d.), (n≥3, transfection experiments).

Source data

Extended Data Fig. 3 Regulation of PLCγ signaling by eDrRTKs.

(a) Top: eDrRTK activates PLCγ. PLCγ catalyzes PIP2 hydrolysis and formation of IP3 and DAG. IP3 interacts with IP3R channels in ER after which they become permeable to Ca2+. In turn, Ca2+ interacts with ORAI channels in the plasma membrane and induces Ca2+ entry from the extracellular space. Bottom: Scheme of the plasmid encoding eDrRTK and GCaMP6m Ca2+ indicator via IRES2. (b) Western blots of phospho-PLCγ, total PLCγ and GAPDH in HEK293 cell lysates and quantification of lane intensities of phospho-PLCγ normalized to GAPDH lane intensities (LIs). (c) Representative HEK293 cells co-transfected with eDrRTKs and GCaMP6m imaged before, during, and after 25 s illumination with 660 nm FR light, and their corresponding calcium flux traces are shown. Red arrows indicate the time when 660 nm light-activation started. After 25 s of 660 nm illumination, inactivating 780 nm light was switched on. Black arrows 1, 2 and 3 indicate time-points when confocal images before illumination, at 660 nm illumination, and after illumination were taken, respectively. 25 µM BV was added to the culture medium in all experiments. Scale bars, 10 µm. Data are presented as mean values, error bars represent standard deviation (s.d.), (n≥3, transfection experiments).

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Unprocessed western blots and statistical source data.

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Unprocessed western blots and statistical source data.

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Leopold, A.V., Thankachan, S., Yang, C. et al. A general approach for engineering RTKs optically controlled with far-red light. Nat Methods 19, 871–880 (2022). https://doi.org/10.1038/s41592-022-01517-z

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