Biosensor construction
Gibson assembly
All cloning, except for site-directed mutagenesis, was conducted using Gibson assembly (NEBuilder HiFi DNA Assembly Master Mix, New England Biolabs)54. All primers were synthesized by BioTeZ. A list of all of the primer sequences is provided in Supplementary Table 5. Primers 1 and 3 were used to cut within the pcDNA3.0 backbone to decrease fragment sizes and increase yields for PCR amplification. All PCR products were obtained using Q5 High-Fidelity DNA Polymerase (New England Biolabs). All constructs were verified by Sanger sequencing (LGC Genomics).
For SP-M 2 R-WT, the cDNA of human WT M 2 R was cloned into pcDNA3.0 and a cleavable signal peptide (SP)55 was cloned N-terminally of WT human M 2 R using primers 1 and 2, 3 and 4, and 5 and 6. To obtain SP-M 2 R-WT-eGFP, eGFP was fused to the C terminus of SP-M 2 R-WT using primers 1 and 7, 3 and 8, and 9 and 10. For the ELISA assay, an HA-tag was cloned N-terminally to the WT receptor, resulting in SP-HA-M 2 R-WT, using primers 3 and 11, and 1 and 12.
Site-directed mutagenesis
Receptor mutants were cloned by introducing an amber stop codon (TAG) at the desired positions by site-directed mutagenesis using the AAscan primer design tool56. A list of the primer sequences is provided in Supplementary Table 6. Mutagenesis was performed using SP-M 2 R-WT, SP-HA-M 2 R-WT and SP-M 2 R-WT-eGFP as templates. Point mutations were introduced by PCR using Thermo Fisher Scientific Phusion High-Fidelity DNA Polymerase (New England Biolabs). PCR products for QuickChange mutagenesis were incubated for 1 h at 37 °C with 1 µl DpnI restriction enzyme (New England Biolabs) before transformation. The resulting mutants are referred to as SP-M 2 RXXXTAG, SP-HA-SP-M 2 RXXXTAG or SP-M 2 RXXXTAG-eGFP throughout. To introduce the mutation disrupting the tyrosine lid closure (Y426A), the primers 13 and 14, and SP-M 2 R175TAG as a template were used, resulting in SP-M 2 R175TAG-Y426A. The point mutation was introduced using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).
Cell culture
HEK-tsA201 (Sigma-Aldrich; referred to as HEK293T cells throughout) cells were cultured in T75 flasks at 37 °C, 5% CO 2 in complete DMEM with 4.5 g l−1 glucose (PAN-Biotech). Culture media was supplemented with 10% (v/v) FBS (Biochrom), 100 U ml−1 penicillin, 100 mg ml−1 streptomycin (Biochrom) and 2 mM L-glutamine (PAN-Biotech). Cells were passaged every 2–3 days when reaching a confluency of 80–90%. For passaging and seeding, the culture medium was aspirated, cells were washed with 5 ml Dulbecco’s PBS solution (Sigma-Aldrich), detached with 2 ml trypsin/EDTA (PAN-Biotech), resuspended in 5 ml DMEM and transferred to a new T75 flask. All cell lines were routinely tested for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza Group) and were not contaminated with mycoplasma. For the qualitative screen of labelled mutants at the accessible extracellular receptor surface, HEK293T cells were seeded on glass-bottomed 8-well µ-slides (Ibidi) at a density of approximately 7 × 104 cells per well in 300 µl DMEM. For single-cell fluorescence microscopy experiments, HEK293T cells were seeded on 24 mm glass coverslips (Paul Marienfeld) in 6-well plates at a density of approximately 2.5–3 × 105 cells per well in 1.5 ml culture medium. Coverslips and 8-well µ-slides were coated with poly-D-lysine (PDL; Sigma-Aldrich; 25 µg ml−1 in PBS) for 30 min at room temperature and washed with PBS twice before seeding cells. For the quantification of labelling efficiency by temporal brightness experiments, HEK-293AD (BioCat; referred to as HEK-AD cells throughout) cells were seeded on uncoated 24 mm glass coverslips in 6-well plates at a density of approximately 3 × 105 cells per well in 1.5 ml culture medium. To determine the cell-surface expression of M 2 R biosensors using a cell-surface enzyme-linked immunosorbent assay (ELISA), 1.6 × 106 HEK293T cells were seeded into a T25 flask and grown for 24 h at 37 °C. For the TRUPATH G-protein activation experiments of WT M 2 receptors, HEK293T cells were seeded into 6-well plates at a density of 3 × 105 cells per well.
For the TRUPATH Gα oA -activation assay of WT M 2 R and each of the seven M 2 R biosensors as well as SP-M 2 R175TAG-Y426A, HEK293T cells were seeded into T75 flasks and grown for 24 h at 37 °C to a confluency of 80–85%.
For the internalization experiments, HEK293T cells were seeded onto glass-bottomed 4-well µ-slides (Ibidi) at a density of approximately 5 × 104 cells per well in 300 µl DMEM.
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