Green-Light-Induced Inactivation of Receptor Signaling Using Cobalamin-Binding Domains

Abstract

Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.

Keywords: cobalamins; optogenetics; photochromism; receptors.

Figure 1

a) Chromophores of main photoreceptor classes (1: p‐coumaric acid, 2: flavins, 3: retinal, 4: tetrapyrroles, 5: AdoCbl). b) Domain structure of CarH with light‐sensitive CBD and DNA binding domain (DBD; HTH=helix‐turn‐helix motif), drawn to scale for CarH of M. xanthus. c) In the dark, CBDs with bound AdoCbl assemble into tetramers. Upon illumination, the 5′‐deoxyadenosyl group is cleaved, and the complex dissociates. d) Fluorescence intensity of mV‐tagged MxCBD, TtCBD, or FKBP expressed in HEK293 cells, with non‐transfected (n.t.) cells as negative control. e) Metabolic activity of HEK293 cells expressing mV‐MxCBD, mV‐TtCBD, or mV‐FKBP and a 5 % DMSO treated control with reduced viability. Mean values ± SEM for three independent experiments each performed in triplicate are given in (d) and (e).

Figure 2

a) CBDs were fused to the ICD of mFGFR1 to engineer receptors that are inactivated by green light. b) Expression of mFGFR1‐MxCBD and mFGFR1‐TtCBD in HEK293 cells supplemented with AdoCbl or CNCbl. c) Activation of the MAPK/ERK pathway (LR=luminescence ratio) by mFGFR1 fused to CBDs or the Fc domain of IgG1 (IgG) and non‐transfected (n.t.) cells in response to red (R; λ=670±5 nm, I=14 μW cm⁻²), green (G; λ=545±5 nm, I=170 μW cm⁻²), or blue (B; λ=470±5 nm, I=200 μW cm⁻²) light. Mean values ± SEM for three to twelve independent experiments each performed in triplicate are given. d, e) Phosphorylation of Erk and mFGFR1‐MxCBD or mFGFR1‐TtCBD in response to green light (0–30 min and after recovery for 60 min in the dark; λ=545±5 nm, I=170 μW cm⁻²).

Figure 3

Embryos a) not injected (n.i.) or injected at the one‐cell stage with b) constitutively active mFGFR1‐IgG (13 pg plasmid), c) AdoCbl (50 fmol), d) mFGFR1‐MxCBD (13 pg plasmid), e) mFGFR1‐MxCBD (13 pg plasmid) and AdoCbl (25 fmol) raised in the dark (D), and f) mFGFR1‐MxCBD (13 pg plasmid) and AdoCbl (25 fmol) raised under green light irradiation (L; 545±5 nm, I=180 μW cm⁻² from 1 to 24 hpf). The images were recorded after 24 (a, c, d) and 30 hpf (b, e, f). g) Quantification of phenotypes (numbers denote the number of embryos).