Bioorthogonal click chemistry-based labelling of proteins in living neuronal cell lines and primary neurons

Developments in microscopy techniques have brought about a demand for new protein labelling methods. Labelling tags should be small and ideally compatible with live-cell imaging. An elegant protein labelling technique that ful�ls these criteria is based on genetic code expansion and bioorthogonal click chemistry. Genetic code expansion allows incorporation of clickable UAAs into proteins of interest which are subsequently directly labelled with small �uorescent dyes using click chemistry. While previous work has mainly focused on easy-to-transfect standard cell lines, recent publications have described applications of this technology in primary neurons. Here, we report a detailed protocol for transfections and bioorthogonal click chemistry-based labelling of intracellular proteins in living primary neurons.


Introduction
In the last two decades, genetic code expansion has emerged as one of the most powerful protein engineering techniques 1,2 .This technology allows incorporation of unnatural (also referred to as noncanonical) amino acids, into proteins in vitro and in vivo.A plethora of custom-designed UAAs with chemically unique side chains have facilitated novel studies in single cells and organisms [3][4][5] .This technology also offers novel avenues for addressing complex neurobiological questions 6 .
In combination with bioorthogonal click chemistry, genetic code expansion can be used for direct protein labelling with small organic dyes [7][8][9][10] .To this aim, clickable UAAs carrying strained alkenes or alkynes, such as trans-cyclooct-2-en-lysine (TCO*-Lys), trans-cyclooct-4-en-lysine (TCO−Lys), strained cyclooctynelysine (SCO−Lys), and bicyclononyne-lysine (BCN−Lys), are incorporated site-speci cally into proteins of interest in response to an in-frame amber stop codon in the corresponding mRNA [11][12][13][14] .Site-speci c UAA incorporation is achieved by amber codon suppression with the help of amber codon suppressor transfer (t)RNA and its cognate aminoacyl-tRNA synthetase (RS).Proteins with UAA are subsequently labelled with uorescent tetrazine dyes in a fast type of bioorthogonal click reaction strain-promoted inverseelectron-demand Diels-Alder cycloaddition (SPIEDAC).This reaction is biocompatible and labelling is done in living cells.What makes this type of labelling particularly attractive is the small size of the labelling tag.Only one native amino acid is exchanged for the UAA in the protein of interest which has minimal steric and functional impact on the protein.As a consequence, dyes are brought as close as possible to the protein of interest.Compared to conventional labelling tags, such as antibodies, and selflabeling tags such as Halo-, SNAP-, CLIP-tags, direct labelling of proteins with tetrazine dyes results in a much smaller linkage error.This makes click chemistry-based protein labelling particularly relevant for super-resolution imaging 8 .In addition, the structure of the protein is minimally perturbed, which makes it particularly attractive for labelling of complex proteins, such as ion channels and receptors, as well as for live-cell imaging studies in general.
Until recently this type of labelling has been mainly used in conventional cell lines 15 .Earlier this year, we have shown that it can be used for labelling of proteins in living neurons, either under exogenous or endogenous promoters 16 .In parallel, others have shown that it can be applied for the labelling of transmembrane proteins in dissociated neurons and slices 17,18 .
Here, we describe a detailed protocol for genetic code expansion and click labelling of intracellular proteins in living neuronal cell lines and primary mouse neurons.We have used neuro lament light chain as a protein of interest, but the protocol can be adjusted for other intracellular or extracellular proteins.As our previous protocols 9,19 deal with the details of genetic code expansion and different click labelling approaches in conventional cell lines, here we report on the optimal and highly e cient conditions for the transfection and click labelling of intracellular proteins in neurons.In addition, we provide a protocol for dual-color pulse-chase click chemistry labeling of two protein populations in living and xed primary neurons.Mouse neuroblastoma x rat neuron hybrid ND7/23 cells (ECACC 92090903), poly-d-lysine (cat.no.P6407), penicillin-streptomycin (cat.no.P0781), paraformaldehyde (PFA; cat.no.158127), and Triton X-100 (cat.no.X100) were purchased from Sigma-Aldrich.

Reagents
Hibernate E medium was purchased from Brain Bits LLC, cat.no.HELF.Electron microscopy grade PFA was purchased from Electron Microscopy Sciences, cat.no.15710.Axial isomer of trans-cyclooct-2-en-l-lysine (TCO*A-Lys) was purchased from Sirius Fine Chemicals, SICHEM, cat.no.SC-8008).100 mM stock solution was made by dissolving TCO*A-Lys in 0.2 M NaOH containing 15% DMSO, and kept at -20° C. Working solution was prepared by diluting the TCO*A-Lys stock 1:4 in 1 M HEPES, and used immediately after.For more details, refer to the references 9 and 19.
For more details on plasmids, cloning and mutagenesis, refer to the reference 16. 13.Right before the medium change, prepare a fresh dilution of TCO*A-Lys as described above (section I, step 9) and pre-warm ND7/23 cell culturing medium to 37° C. 25.Alternatively, if cell-impermeable tetrazine-dyes are to be used for labeling, x the cells after labeling and washing (section I, steps 16-23) with 4% PFA diluted in PB for 15 min at RT. Rinse the cells 3x with PBS and permeabilize with 0.1% Triton X-100 diluted in PBS for 10 min at RT. Incubate for 10 min at 37° C with the cell-impermeable tetrazine-dye diluted to the nal concentration of 0.5-2.5 µM in PBS.ATTO488tz dye works well for labeling in xed cells, although with a higher non-speci c background staining in comparison to live cell labeling with cell-permeable dyes.Aspirate the dye, rinse 3x with PBS and incubate with PBS on a shaker at RT for 20-30 min.Keep at 4° C until immunocytochemistry staining or microscopy.

II. Bioorthogonal click chemistry labeling of proteins in primary neurons
Neuron seeding and transfection Day 0: Neuron seeding 1. Pre-coat eight-well Lab-Tek II chambered coverglasses with 20 µg/ml solution of poly-d-lysine in ddH2O for 2 h at RT. Wash the chambered coverglasses three times with ddH2O and allow to dry under a sterile hood.Pre-incubate coated coverglasses for at least 30 min at 37° C with neuron culturing medium consisting of Neurobasal Plus (NB Plus) medium with the addition of 2% B27 Plus and 1% PS (referred to as NB Plus +).
2. Thaw primary mouse cortical neurons (MCNs) from C57BL/6 embryonic day 17 according to the manufacturer's recommendation and seed them at the density of 90,000-110,000 cells, in 500 µl of medium per well.Alternatively, fresh primary mouse or rat neurons can be prepared according to other established protocols of your choice.second population labeling after xation, we used cell-impermeable ATTO488-tz.Aspirate the dye, rinse 3x with PBS and incubate with PBS on a shaker at RT for 20-30 min.Keep at 4° C until immunocytochemistry staining or microscopy.

Troubleshooting
Section I, step 5 & section II, step 6: depending on the experimental design, different variants the NES PylRS/tRNA CUA Pyl containing plasmid can be used.To increase e ciency of UAA incorporation, we obtained good results using codon-optimized NES PylRS/tRNA CUA Pyl and eRF1 E55D containing plasmids.
These plasmids are listed under reagents.More details can be found in 16 .
Section II, step 4: our transfection protocol is established based on the published work 20 , but different transfection conditions and reagents could be used.
Section II, step 24: incubation up to 10 h after click labeling reduces the non-speci c background labeling.
More details can be found in 16 .
Section II, step 28: please note that the duration of the 2 nd UAA incubation step can be adjusted according to the desired experimental design.In the manuscript associated with this protocol, we have tried 3h, 1 days and 2 days.More details can be found in 16 .Overall, single-color labeling procedure from neuron seeding to xation or live imaging takes 10 or 11 days, while dual color labeling procedure usually takes 12 days.

Anticipated Results
Figure 1 shows representative images of click-labeled neuro lament light chain (NFL) in ND7/23 cells and primary neurons.More examples can be found in 16 .

Figures
Figure 1 Representative confocal images of ND7/23 cells and primary neurons expresing clickable NFL(K363TAG)-FLAG together with NES PylRS/tRNA and NFM.Cells and neurons were seeded and transfected as described in the protocol.After incubation with TCO*A-Lys, they were labeled live with 5µM of cell-permeable SiR-tz for 10 min at 37° C, xed and ammunostained with anti FLAG primary antibody, followed by an Alexa Fluor 488-conjugated secondary antibody.

2 . 3 . 5 . 6 . 7 . 8 .
Standard cell culture equipment, facility designated for genetic engineering work (please note that depending on your local regulations different biosafety regulations might apply), and a uorescent light microscope of choice.Procedure I. Bioorthogonal click chemistry labeling of proteins in ND7/23 neuroblastoma cells Day 0: ND7/23 cell seeding 1. Pre-coat eight-well Lab-Tek II chambered coverglasses with 10 µg/ml solution of poly-D-lysine in double-distilled water (ddH 2 O) for a minimum of 4 h at room temperature (RT).Wash the chambered coverglasses three times with ddH 2 O and allow to dry under a sterile hood.Collect the cells from the culturing plate and seed at a density of 25,000 cells per well in 250 µl of ND7/23 cell culturing medium which contains high-glucose Dulbecco's Modi ed Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (inactivated by incubation at 56 °C for 30 min), 1% penicillin-streptomycin (PS), 1% sodium pyruvate and 1% L-glutamine.To ensure optimal conditions for cell growth, use cell passages 3 to 15 for these types of experiments.Culture seeded cells overnight at 37° C, 5% CO 2 .Day 1: ND7/23 cell transfection 4. Pre-warm Opti-MEM™ I Reduced Serum Medium to 37° C. Prepare transfection mix with a DNA/Lipofectamine 2000 ratio of 1 µg:2.4µl.A master mix can be prepared for the transfection of multiple wells.For one well of an eight-well Lab-Tek II chamber prepare the following: a. Tube 1: Add 12.5 µl of Opti-MEM.Add 0.25 µg of NFL K363TAG -FLAG, 0.25 µg of NES PylRS/tRNA CUA Pyl and 0.125 µg of NFM plasmid DNA to the Opti-MEM.b.Tube 2: Add 12.5 µl of Opti-MEM.Add 1.5 µl of Lipofectamine 2000 transfection reagent to the Opti-MEM.Vortex the tubes gently for 10 seconds, then centrifuge brie y to spin down.Incubate 5 minutes (min) at RT. Add the contents of tube 1 to the tube 2. Vortex the tube gently for 10 seconds, then centrifuge brie y to spin down.Incubate 20 min at RT. 9.During the 20 min-long incubation prepare a 1:4 dilution of TCO*A-Lys in 1 M HEPES.For one well of an eight-well Lab-Tek II chamber mix 1.875 µl of 1 M HEPES and 0.625 µl of 100 mM TCO*A-Lys stock solution.*A master mix can be prepared if TCO*A-Lys is to be added to multiple wells.10.Take the cells out of the incubator.Add 25 µl of the transfection mix dropwise to the culturing medium.Handle up to two Lab-Tek II chambered coverglasses at a time, to prevent the culturing medium from cooling down.11.Add 2.5 µl of TCO*A-Lys dilution in a corner of the well.Move the Lab-Tek II chambered coverglass up and down, left and right to resuspend the TCO*A-Lys.The nal concentration of TCO*A-Lys is 250 µM.*After the addition of TCO*A-Lys dilution, the pink color of the medium will become slightly more red/orange due to the change in pH 12. Incubate the cells 6 h at 37° C, 5% CO2.

Day 3 and day 7 : 5 . 6 .
Medium change 3. Pre-warm the NB Plus + medium at 37° C. Aspirate 200 µl of the medium from each well and add 250 µl of fresh warm NB Plus + medium.When changing the medium on day 7, collect the aspirated medium in tubes and keep at 4° C for further use as conditioned medium (CM).Day 8: Transfection 4. Transfect MCNs using Lipofectamine 2000 and a DNA/Lipofectamine 2000 ratio of 1 µg:2.4µl as described below.Prepare the transfection medium A by adding 1% of PS to Neurobasal Plus medium.Prepare the transfection medium B by adding 1% of PS and 4% of B27 Plus to Neurobasal Plus medium.Warm up both transfection media to 37° C. Prepare the transfection mix.A master mix can be prepared for the transfection of multiple wells.For one well of an eight-well Lab-Tek II chamber prepare the following: a. Tube 1: Add 50 µl of transfection medium A. Add 0.5 µg of NFL K363TAG -FLAG, 0.5 µg of NES PylRS/tRNA CUA Pyl and 0.25 µg of NFM plasmid DNA to the transfection medium.b.Tube 2: Add 50 µl of transfection medium A. Add 3 µl of Lipofectamine 2000 transfection reagent to the transfection medium.

Section I:
Bioorthogonal click chemistry labeling of proteins in ND7/23 neuroblastoma cells Coating and preparation of Lab-Tek chambers takes around 4 h, ND7/23 cell seeding takes around 20 min, transfection around 45 min, live-cell click chemistry labeling around 30 min, xation 20 min, and xed-cell click chemistry labeling around 1 h.Overall, the procedure from cell seeding to cell xation takes 3 days.Section Bioorthogonal click chemistry labeling proteins in primary neurons Coating and preparation of Lab-Tek chambers takes around 3h, neuron seeding takes around 1h, transfection around 1 h, live-cell click chemistry labeling around 30 min, xation 20 min, and xed-cell click chemistry labeling around 1 h.
14. Aspirate the transfection mix-containing medium from cells and add 250 µl of warm ND7/23 cell culturing medium.Add TCO*A-Lys dilution as described above (section I, step 11).Lys from cells by aspirating the medium and rinsing 2x with warm cell culturing medium.Add 250 µl of culturing medium per one well of an eight-well Lab-Tek II chamber.
19. Prepare a 5 µM tetrazine-dye dilution in warm culturing medium.For example, silicon rhodaminetetrazine (SiR-tz) dye performs well for live cell labeling of intracellular proteins, and we use it most