Spatio-temporally precise activation of engineered receptor tyrosine kinases by light

Abstract

Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour.

Keywords: aureochrome; cell signalling; light‐oxygen‐voltage (LOV)‐sensing domain; optogenetics; synthetic biology; synthetic physiology.

Figure 1. Selection of LOV domains and expression in mammalian cells

1. Domain structure of light-sensing proteins from which LOV domains (highlighted with asterisk) were excised (AtPH1 and AtPH2, Arabidopsis thaliana phototropin 1 and 2; CrPH, Chlamydomonas rheinhardtii phototropin; NcVV, Neurospora crassa vivid; NcWC1, N. crassa white collar 1; RsLP, Rhodobacter sphaeroides ATCC 17025 light-sensing protein; VfAU1, Vaucheria frigida aureochrome1). In these proteins, LOV domains regulate a variety of effector domains (STK, serine/threonine kinase; DB, DNA-binding domain). To test for expression and influence on cell viability in mammalian cells, LOV domains optimized for mammalian codon usage were fused to the fluorescent protein mVenus (mV).

2. Fluorescence intensity measurements of human embryonic kidney (HEK) 293 cells transfected with mVenus-LOV domain fusions.

3. Viability of HEK293 cells transfected with mVenus-LOV domain fusions.

4. Fluorescence intensity measurements of Chinese hamster ovary (CHO) K1 cells transfected with mVenus-LOV domain fusions.

5. Viability of CHO K1 cells transfected with mVenus-LOV domain fusions.

Data information: For (B–E): fluorescence and viability were quantified 16–18 h after transfection. Data were normalized to mV fused to the small, robustly folding FK506 binding protein (FKBP). Mean values ± SD for three independent experiments each performed in quadruplicates are shown.

Figure 2. Design and function of mFGFR1-LOV domain chimeric receptors

1. Receptor tyrosine kinases such as mFGFR1 consist of the extracellular ligand-binding domain (LBD), single-span transmembrane domain (TMD) and intracellular domain (ICD) [kinase domain (KD) and a C-terminal tail domain (CTD)]. In mFGFR1-LOV domain chimeras, only the ICD is retained to render the protein insensitive to endogenous ligand. The ICD is attached to the membrane using a myristoylation domain (MYR) and LOV domains are incorporated at the ICD C-terminus.

2. MAPK/ERK pathway activation in response to blue light for HEK293 cells that were transfected with chimeric proteins of mFGFR1-ICD and LOV domains. Activation is expressed as induction of a luciferase reporter gene. imFGFR1 is activated by the small molecule dimerizer AP20187.

3. MAPK/ERK pathway activation in response to blue, green and red light for HEK293 cells that were transfected with imFGFR1, Opto-mFGFR1 (mFGFR1-VfAU1-LOV) or kinase dead Opto-mFGFR1 (Y271F, Y272F).

Data information: For (B) and (C): 24 h after transfection, cells were stimulated with light for 8 h followed by detection of luciferase. Light intensity was 1.7–2.5 μW/mm². Mean values ± SEM for four to 16 independent experiments each performed in triplicates are shown.

Figure 3. Analysis of Opto-mFGFR1 dimerization and sites required for MAPK/ERK pathway activation

1. MAPK/ERK pathway activation in response to blue light for HEK293 cells that were transfected with imFGFR1, Opto-mFGFR1 or genes with R195E substitution. This substitution prevents functional KD dimerization.

2. Chemical cross-linking reveals light-induced dimerization of Opto-mFGFR1 in M38K^Opto-mFGFR1 cells. Cells were stimulated with blue light for 15 min.

3. Single-site or multi-site substitutions test phosphorylation site and coupling site requirements of Opto-mFGFR1. MAPK/ERK pathway activation in HEK293 cells that were transfected with Opto-mFGFR1 harbouring Tyr substitions at positions 81 (463), 201 (583), 203 (585), 348 (730), 384 (766) and 447 (3) or combined substitutions to Ala (‘4A’) at positions K37, P40, L41 and R43 (K419, P422, L423 and R425; numbering is relative to start methionine of Opto-mFGFR1; corresponding positions in mFGFR1 or VfAU1-LOV are given in parentheses). These residues were previously shown to be phosphorylated (Tyr) or required for association of FRS2 (LysProLeuArg).

Data information: For (A) and (C): 24 h after transfection, cells were stimulated with blue light for 8 h followed by detection of luciferase. Mean values ± SEM for two to 16 independent experiments each performed in triplicates are shown. For (A–C): Light intensity was ˜2.5 μW/mm².

Figure 4. Analysis of VfAU1-LOV photoadduct lifetime, additional receptor tyrosine kinases and alternative LOV domains

1. MAPK/ERK pathway activation in response to blue light for HEK293 cells that were transfected with Opto-mFGFR1 or Opto-mFGFR1 with I472V substitution. This substitution reduces VfAU1-LOV photoadduct lifetime.

2. MAPK/ERK pathway activation in response to blue light for HEK293 cells that were transfected with chimeric proteins of hEGFR1-ICD or hRET-ICD and VfAU1-LOV.

3. MAPK/ERK pathway activation in response to blue light for HEK293 cells that were transfected with chimeric proteins of mFGFR1-ICD and NgPA1-LOV or OdPA1-LOV.

Data information: 24 h after transfection, cells were stimulated with blue light for 8 h followed by detection of luciferase. Light intensity was ˜2.5 μW/mm². Mean values ± SEM for three to 16 independent experiments each performed in triplicates are shown.

Figure 5. Activation of cellular signalling with temporal precision

1. Phosphorylation of Opto-mFGFR1 and ERK1/2 in human malignant pleural mesothelioma cells (M38K^Opto-mFGFR1, SPC212^Opto-mFGFR1) in response to 1 min of blue light followed by indicated duration of darkness.

2. Phosphorylation of AKT and PLCγ1 in SPC212^Opto-mFGFR1 in response to 1 min of blue light followed by indicated lengths of darkness.

3. mFGFR1-VfAU1-LOV and ERK1/2 phosphorylation in hBE^Opto-mFGFR1 cells in response to different durations of blue light.

Figure 6. Activation of cellular signalling with spatial precision

1. Spatially confined ERK1/2 phosphorylation in SPC212^Opto-mFGFR1 cells. A illumination pattern of 16 equidistant circles was produced using a pinhole template. Stars mark circles in corners. Dashed line marks a single circle. Cells were stimulated with blue light for 5 min followed by fixation and staining. Scale bar: 2 mm.

2. Spatially confined ERK1/2 phosphorylation in hBE^Opto-mFGFR1 cells. A illumination pattern of two circles was produced using a pinhole template. Star marks the center of one circle, dashed line marks the other circle. Cells were stimulated with blue light for 5 min followed by fixation and staining. Scale bar: 5 mm.

3. Spatially confined MAPK-dependent gene transcription in HEK293 cells. Thirty-four hours after transfection, cells stimulated with light for 8 h followed by live-cell detection of luciferase. Scale bar: 10 mm.

Data information: All images are unprocessed raw images. Light intensity was ˜2.5 μW/mm².

Figure 7. Optical control of cell behaviour

1. Percentage of DNA synthesis in control and M38K^Opto-mFGFR1 cells in response to blue light or FGF2 ligand. Cells were stimulated with blue light for 1 h. Proliferation was analysed after 26 h. Mean values ± SEM for 15–20 micrographs with 50–300 cells per micrograph from two independent experiments are shown.

2. Morphology changes in control and M38K^Opto-mFGFR1 cells in response to blue light or FGF2 ligand. Cells were stimulated with blue light for 1 h. Morphology was analysed after 24 h. Mean values ± SEM for 60 individual cells from two independent experiments are shown.

3. Representative images for (B). Scale bar: 40 μm.

4. Sprouting in control and hBE^Opto-mFGFR1 cells in response to blue light or VEGFA ligand. Cells were stimulated with blue light for 5 min every 10 min during 10 h followed by analysis or morphology. Mean values ± SEM for 8–11 spheres per group in three independent experiments are shown.

5. Representative images for (D). Scale bar: 100 μm.

Data information: Control cells were infected with mCherry. P < 0.05 for stimulated versus unstimulated cells (One-way ANOVA with Tukey's post-hoc test); n.s. for light stimulation versus FGF2/VEGFA stimulation; n.s. for unstimulated control cells versus unstimulated Opto-mFGFR1 cells. Light intensity was ˜2.5 μW/mm².