Site-Specific L-Anap Incorporation by Genetic Code Expansion for Minimally Perturbative Imaging of TDP-43 in Mammalian Cells

Abstract

Fluorescent protein tags are widely used to investigate protein localization and dynamics, but their relatively large size can perturb protein structure and function. In contrast, genetic code expansion offers a minimally disruptive alternative by enabling site-specific incorporation of non-canonical amino acids, such as L-Anap (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid). Here, we describe a protocol for labeling TDP-43 with the intrinsically fluorescent non-canonical amino acid L-Anap. The protocol covers plasmid transfection, L-Anap incorporation, removal of unincorporated L-Anap, immunofluorescence staining, and live-cell imaging. The resulting L-Anap-labeled TDP-43 protein can be visualized directly without additional chemical labeling or the attachment of a bulky terminal tag. Overall, this protocol provides a versatile platform for investigating protein localization and dynamics while minimizing perturbations to protein structure and function.

Key features

• This protocol enables site-specific protein labeling while minimizing disruption to the structure and function of the protein of interest.

• The protocol allows labeling at selected internal amino acid positions and is therefore not restricted to the N- or C-terminus of the protein.

• The protocol is compatible with both live-cell and fixed-cell imaging.

Keywords

Neurodegeneration; Amyotrophic lateral sclerosis (ALS); Genetic code expansion; L-Anap; TDP-43

Graphical overview

Background

TAR DNA-binding protein 43 (TDP-43) is a predominantly nuclear RNA-binding protein that regulates multiple aspects of RNA metabolism [1,2]. Nuclear depletion of TDP-43 accompanied by the formation of cytoplasmic TDP-43 inclusions is a defining pathological feature of amyotrophic lateral sclerosis (ALS) and several related neurodegenerative disorders [3,4]. However, faithfully visualizing TDP-43 remains challenging because its aggregation behavior is closely linked to its domain organization and proteinopathy aggregation. In particular, the low-complexity intrinsically disordered region at the C-terminus contributes to protein-protein interactions, liquid-liquid phase separation, and pathological aggregation [5].  Traditional fluorescent tags, such as GFP and mCherry, can alter the natural behavior of TDP-43, and tagged constructs frequently require modifications such as deletion of the nuclear localization signal (NLS) [6,7].

To resolve this problem, we used the genetic code expansion (GCE) technique, which enables minimally disruptive fluorescent labeling of TDP-43 in mammalian cells. GCE enables the site-specific incorporation of non-canonical amino acids into proteins in living cells by reassigning the amber stop codon (TAG), using an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair [8]. Compared with introducing a fluorescent tag, GCE introduces only a single non-canonical amino acid at a selected residue [8-11], reducing structural and functional perturbations associated with protein labeling. L-Anap (3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid) is an intrinsically fluorescent and environmentally sensitive non-canonical amino acid that can be directly detected without an additional chemical-labeling reaction [8,10]. When an appropriate incorporation site is selected outside essential functional motifs, GCE-mediated L-Anap incorporation can therefore provide a minimally disruptive approach for monitoring protein localization and dynamics while better preserving native protein structure, interactions, and function.

Here, we describe a GCE-based protocol for minimally disruptive fluorescent labeling of TDP-43 in mammalian cells. The codon for residue V100 of TDP-43 is replaced with an amber codon, and the resulting TDP-43-V100TAG construct is co-expressed with the pAnap orthogonal translation system in the presence of L-Anap. The incorporated L-Anap allows direct visualization of TDP-43 without attaching a large fluorescent protein or disrupting its nuclear localization signal. This protocol covers construction of the V100TAG TDP-43-mutant plasmid, L-Anap incorporation, removal of unincorporated L-Anap, fixed-cell immunofluorescence, and live-cell imaging. TDP-43-Anap retains predominantly nuclear localization under basal conditions and enables visualization of stress-induced cytoplasmic TDP-43 accumulation. The method therefore provides a minimally perturbative platform for investigating TDP-43 localization and dynamics in both live and fixed cells.

Materials and reagents

Biological materials

1. HeLa cells
2. Primary mouse cortical neurons
3. DH5α component E. coli.
4. pAnap plasmid encoding the orthogonal Anap aminoacyl-tRNA synthetase/tRNA pair (gift from the Peter G. Schultz laboratory [8])
5. pRK5-TDP-43 (generated in-house) 
6. pRK5-TDP-43-V100TAG (generated in-house [12])

Reagents

1. L-Anap trifluoroacetate salt (Cayman Chemical, catalog number 15436)
2. Q5^® Site-Directed Mutagenesis Kit (NEB, catalog number E0554S)
3. DMEM/F-12 medium (Corning, catalog number 10-013-CV)
4. Fetal bovine serum (Gibco, catalog number A5256801)
5. Lipofectamine 2000 (Thermo Fisher Scientific, catalog number 11668030)
6. Lipofectamine 3000 with P3000 reagent (Thermo Fisher Scientific, catalog number L3000015)
7. FluoroBrite DMEM (Thermo Fisher Scientific, catalog number A1896701)
8. Opti-MEM medium (Thermo Fisher Scientific, catalog number 31985070)
9. Dulbecco's phosphate-buffered saline
10. Protease inhibitor cocktail (Millipore Sigma, catalog number P8340)
11. Paraformaldehyde (MilliporeSigma, catalog number 158127)
12. Immunofluorescence blocking buffer (Cell Signaling Technology, catalog number 12411)
13. Triton X-100 (MilliporeSigma, catalog number X100)
14. Mouse anti-human G3BP1 antibody (BD Biosciences, catalog number 611126)
15. Rabbit anti-TDP-43 antibody (Proteintech, catalog number 10782-2-AP)
16. Mouse anti-human-specific TDP-43 monoclonal antibody (Proteintech, catalog number 60019-2-Ig; optional neuronal adaptation)
17. Chicken anti-beta-III-tubulin/Tuj1 antibody (GeneTex, catalog number GTX85469; optional neuronal adaptation)
18. Donkey anti-mouse Alexa Fluor 555 antibody (Thermo Fisher Scientific, catalog number A-31570)
19. Donkey anti-rabbit Alexa Fluor 488 antibody (Thermo Fisher Scientific, catalog number A-21202)
20. Donkey anti-chicken Alexa Fluor 647 antibody (Thermo Fisher Scientific, catalog number A78952; optional neuronal adaptation)
21. HBSS, 0.25% trypsin, DNase I, and DMEM (optional neuronal isolation)
22. Poly-D-lysine (Gibco, catalog number A3890401; optional neuronal adaptation)
23. Neurobasal medium (Gibco, catalog number 21103049; optional neuronal adaptation)
24. B-27 supplement (Gibco, catalog number 17504044; optional neuronal adaptation)
25. GlutaMAX supplement (Gibco, catalog number 35050061; optional neuronal adaptation)
26. Penicillin-streptomycin (Gibco, catalog number 15140163; optional neuronal adaptation)

Solutions

1. HeLa complete growth medium (see Recipes)
2. 20 µM L-Anap HeLa labeling medium (see Recipes)
3. 0.1% Triton X-100 immunofluorescence blocking/permeabilization buffer (see Recipes)
4. Neuronal plating medium (optional; see Recipes)
5. Neuronal maintenance medium (optional; see Recipes)
6. 10 µM L-Anap neuronal labeling medium (optional; see Recipes)

Recipes

1. HeLa complete growth medium (100 mL)

2. L-Anap HeLa labeling medium (10 mL)

Note: Dissolve 3.86 mg of L-ANAP trifluoroacetate salt in anhydrous DMSO and bring the final volume to 1 mL (10 mM stock). Mix until completely dissolved. L-Anap stock solution can be stored at −20 °C for short-term use and should be stored at −80 °C for long-term storage. Protect the aliquot from light and avoid repeated freeze-thaw cycles. 

3. Immunofluorescence blocking/permeabilization buffer (10 mL)

4. Neuronal plating medium (100 mL; optional)

5. Neuronal maintenance medium (100 mL; optional)

6. L-Anap neuronal labeling medium (10 mL; optional)

Note: Dissolve 3.86 mg of L-ANAP trifluoroacetate salt in anhydrous DMSO and bring the final volume to 1 mL (10 mM stock). Mix until completely dissolved. L-Anap stock solution can be stored at −20 °C for short-term use and should be stored at −80 °C for long-term storage. Protect the aliquot from light and avoid repeated freeze-thaw cycles. 

Laboratory supplies

1. Sterile cell-culture plates and dishes appropriate for the selected assay 
2. 35 mm glass-bottom dishes for live-cell imaging
3. Poly-D-lysine-coated culture plates for primary neurons
4. Microscope-compatible coverslips for fixed-cell imaging

Equipment

1. Class II biological safety cabinet
2. Humidified mammalian cell incubator set to 37 °C and 5% CO₂
3. Leica SP8 confocal microscope
4. Routine cell-culture microscope and cell-counting device

Procedure

A. Site selection and construction of the TDP-43 TAG-mutant plasmid

1. Review the full-length human TDP-43 sequence and annotated features in UniProt and inspect its predicted structure using AlphaFold. Exclude residues within annotated protein domains, known interaction interfaces, and other functionally important or highly conserved sites whenever possible. Prioritize solvent-exposed residues in flexible linker regions and avoid residues predicted to contribute to secondary structure, the hydrophobic core, interdomain packing, or stabilizing interaction networks. 
2. For the construct described in this protocol, select V100 as the L-Anap incorporation site. V100 lies outside the annotated folded domains of TDP-43 and was selected to minimize the predicted effects of residue replacement on protein folding and function.
3. Use NEBaseChanger to design a pair of mutagenic primers that replace the codon encoding V100 with the amber stop codon (TAG). Enter the complete TDP-43 cDNA, specify the desired codon substitution, and record the recommended primer sequences and annealing temperature.
4. Set up a 25 µL amplification reaction containing 12.5 µL of Q5 Hot Start High-Fidelity 2x Master Mix, 1.25 µL each of 10 µM forward and reverse primers, 1 µL of pRK5-TDP-43 template (1 ng), and 9 µL of nuclease-free water. Perform PCR using the annealing temperature recommended by NEBaseChanger and the extension time specified by the Q5 Site-Directed Mutagenesis Kit for the plasmid length.
5. For the KLD reaction, combine 1 µL of the PCR product, 5 µL of 2x KLD Reaction Buffer, 1 µL of 10x KLD Enzyme Mix, and 3 µL of nuclease-free water. Incubate for 5 min at room temperature.
6. Transform 5 µL of the KLD reaction into 50 µL of DH5⍺ Competent E. coli. Recover the cells and plate them on ampicillin-containing agar, following the kit manufacturer's instructions.
7. Pick individual colonies, extract plasmid DNA, and confirm the V100TAG substitution by Sanger sequencing. Sequence across the complete TDP-43 coding region to exclude unintended mutations before using the plasmid for L-Anap incorporation experiments.

B. Culture and transfect HeLa cells for Anap incorporation

1. Maintain HeLa cells at 37 °C and 5% CO₂ in HeLa complete growth medium.
2. Seed cells 24 h before transfection in the vessel required for the downstream assay. For live-cell imaging, seed approximately 1.2 × 10⁵ cells in 2 mL of complete growth medium in each 35 mm glass-bottom dish. For fixed-cell imaging, seed approximately 1 x 10⁵ cells in 1 mL of complete growth medium onto a 20 mm coverslip in a 12-well plate. Adjust the initial seeding density as needed, based on the measured growth rate, passage number, and transfection-associated growth inhibition in HeLa cells.
3. For each sample, combine 1.1 μg of pAnap with 0.9 μg of the sequence-verified TDP-43 TAG-mutant plasmid, corresponding to an approximate pAnap:target-plasmid mass ratio of 1.2:1 and a total plasmid DNA amount of 2 μg.
4. Dilute the total plasmid DNA in 100 μL Opti-MEM and mix gently. In a separate tube, dilute 4 μL Lipofectamine 2000 in 100 μL Opti-MEM. Use 2 μL Lipofectamine 2000 per 1 μg total DNA. 
5. Combine the diluted DNA and diluted Lipofectamine 2000, mix gently, and incubate for 15 min at room temperature.
6. Add the complexes evenly to the cells and return the culture to 37 °C and 5% CO₂.
7. At 4-6 h after transfection, replace the medium with fresh HeLa complete growth medium containing 20 μM L-Anap. Incubate for 20-24 h before imaging.
8. After the 20-24 h incorporation period, aspirate the labeling medium and wash the cells three times with DPBS to remove extracellular L-Anap. After washing, add fresh complete growth medium and incubate the cells for 1 h. Replace the medium with fresh complete growth medium again, incubate for an additional 1 h, and then proceed with live-cell imaging or fix the cells with 4% paraformaldehyde.

C. Induce oxidative stress and prepare fixed cells for immunofluorescence

1. Note: Perform all immunofluorescence procedures under reduced-light conditions and protect the samples from light whenever possible to minimize photobleaching of Anap fluorescence.
2. Seed HeLa cells onto a 20 mm coverslip in a 12-well plate. Label TDP-43 with L-Anap as described in procedure B.
3. Prepare matched unstressed and stressed cultures after the 2 h L-Anap washout period.
4. Treat the stressed cultures with 250 μM sodium arsenite for 1 h at 37 °C and 5% CO₂. Maintain the unstressed controls in the corresponding complete medium for the same interval.
5. Wash each culture with DPBS.
6. Fix the cells with 4% paraformaldehyde for 15 min at room temperature.
7. Remove fixative according to institutional hazardous-waste procedures and wash with DPBS three times. 
8. Permeabilize and block the fixed cells for 1 h in immunofluorescence blocking buffer containing 0.1% Triton X-100.
9. Incubate fixed cell samples with mouse anti-human G3BP1 antibody (BD Biosciences, catalog number 611126) diluted 1:500 and rabbit anti-TDP-43 antibody (Proteintech, catalog number 10782-2-AP) diluted 1:1000, as appropriate, overnight at 4 °C. The following day, wash the cells three times with PBS for 10 min per wash.
10. Dilute the species-appropriate Alexa Fluor–conjugated secondary antibody 1:1000 in PBS and incubate the cells for 1 h at room temperature, protected from light. Wash the cells five times with PBS for 10 min per wash while protecting them from light.
11. Acquire Anap (405 nm) and immunofluorescence channels on the Leica SP8 confocal microscope using settings that avoid saturation and spectral bleed-through. Keep settings identical across matched samples (Figure 1).

D. Perform live-cell imaging

1. Seed HeLa cells on 35 mm glass-bottom dishes, transfect with pAnap plus the TDP-43 TAG-mutant plasmid, label with 20 μM L-Anap for 24 h, wash three times with DPBS, and incubate in fresh medium as described above.
2. Replace the culture medium with FluoroBrite DMEM and place the dish in a microscope stage incubator maintained at 37 °C and 5% CO₂. For stress-induced live-cell imaging, treat the cells with 250 μM sodium arsenite for 1 h on the microscope before image acquisition.
3. Identify cells with non-saturated Anap fluorescence and expected localization.
4. Export raw image stacks and acquisition metadata without applying nonlinear display adjustments.

E. Anap incorporation in primary mouse cortical neurons

1. At DIV0, dissect embryonic mouse cortex into ice-cold HBSS under the approved animal protocol.
2. Digest the tissue in 0.25% trypsin containing 0.1 mg/mL DNase I for 20 min at 37 °C.
3. Stop the digestion and wash the tissue twice with neuronal plating medium consisting of DMEM supplemented with 10% FBS.
4. Dissociate the tissue to a single-cell suspension, wash twice, and resuspend the cells in neuronal plating medium.
5. Seed 1 × 10⁵ to 2 × 10⁵ cells per well onto each poly-D-lysine-coated 20 mm coverslip in a 12-well plate. 
6. After 3-4 h, replace the plating medium with neuronal maintenance medium.
7. At DIV5, prepare the transfection mixture for each well. For each sample, combine 0.54 μg of pAnap with 0.45 μg of the sequence-verified TDP-43 TAG-mutant plasmid, corresponding to an approximate pAnap: target-plasmid mass ratio of 1.2:1. The total plasmid DNA amount is 1 μg per well, and the combined Opti-MEM volume is 100 μL per well. In one tube, combine the pAnap and TDP-43 TAG-mutant plasmids with 2 μL of P3000 reagent, and dilute the mixture to 50 μL with Opti-MEM. In a separate tube, dilute 2 μL of Lipofectamine 3000 to 50 μL with Opti-MEM. 
8. Combine the diluted DNA/P3000 mixture with the diluted Lipofectamine 3000, mix gently, incubate for 15 min at room temperature, and then add the complexes to the neurons.
9. After overnight incubation, at DIV6, replace one-half of the medium with fresh neuronal maintenance medium containing L-Anap to reach a final concentration of 10 micromolar L-Anap.
10. Continue incubation for 2 additional days. At DIV8, replace one-half of the medium each day with fresh neuronal maintenance medium containing 10 micromolar L-Anap to maintain the L-Anap supply.
11. Wash out extracellular L-Anap and perform stress treatment, fixation, immunostaining, or live imaging using the corresponding HeLa workflow, adjusting only culture-format-dependent volumes. Include anti-TDP-43 staining and anti-Tuj1 staining when confirming neuronal expression(Figure 2).

Data analysis

Process and analyze all fluorescence images using Fiji/ImageJ. Apply identical processing and display parameters to all images within the same experiment. 

Validation of protocol

This protocol has been validated in the following published article and unpublished experiments:

• Hao Chen et al. [12] Non-canonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy. eLife 

General notes and troubleshooting

General notes

1. L-Anap stock solution should be stored at −80 °C for long-term storage. It may be stored at −20 °C for short-term use, preferably for no longer than one month. Aliquot the stock solution and avoid repeated freeze–thaw cycles.
2. Western blotting can be used to verify the expression and expected molecular weight of the Anap-labeled protein of interest (POI).
3. Cell condition is critical for successful Anap labeling. Use healthy cells with normal morphology and growth characteristics, and routinely confirm that the cultures are free of mycoplasma contamination.
4. Avoid excessive cell confluence during live-cell imaging. A cell confluence of approximately 50%–60% at the time of imaging is recommended.

Troubleshooting

Problem 1: Expression of the POI is detected by western blotting, but no Anap-positive cells are observed by immunofluorescence or live-cell imaging.

Possible cause: The 405 nm excitation laser power is insufficient to detect the Anap fluorescence.

Solution: Gradually increase the 405 nm laser power and optimize the detector gain or emission collection settings. Use identical acquisition settings for the experimental and negative-control groups and avoid excessive excitation that may cause rapid Anap photobleaching.

Problem 2: Fluorescence is also detected in the negative-control group.

Possible cause: Extracellular or unincorporated L-Anap has not been removed completely.

Solution: Wash the cells thoroughly with DPBS and extend the incubation period in fresh, L-Anap-free complete growth medium. If necessary, perform an additional medium replacement during the washout period before imaging.

Problem 3: No clear fluorescence signal from the labeled POI is observed, and western blotting also fails to detect the expected full-length protein.

Possible cause: The L-Anap stock may have lost activity, or the selected amber-codon incorporation site may not support efficient Anap incorporation or full-length protein expression.

Solution: Repeat the experiment using a freshly prepared or properly stored L-Anap stock. If full-length protein expression remains undetectable, select and validate an alternative labeling site in the POI.

Acknowledgments

This work was supported by the National Institutes of Health (NIH) under grant numbers NS074324, NS089616, NS110098, and NS128494; the Walder Foundation; the Packard Center for ALS Research at Johns Hopkins; and the Maryland Stem Cell Research Fund. We thank Peter G. Schultz of Scripps Research for providing the pAnap plasmid and Philip Wong of Johns Hopkins University for providing the inducible TDP-43 knockout (iTDPKO) mouse embryonic stem cell line. We also thank the members of the Wang laboratory for their helpful discussions and suggestions. The graphical abstract was created with BioRender.com. This protocol was developed primarily based on the study by Hao Chen et al. [12].

Competing interests

The authors declare no financial or non-financial competing interests.

Ethical considerations

No animal or human subjects were used during this study.

References

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Figure Legends

Figure 1: Fluorescence imaging of TDP-Anap and stress granules under basal and stress conditions. Representative fluorescence images of HeLa cells expressing TDP-Anap under untreated (NT) conditions or following sodium arsenite treatment (SA). Intrinsic TDP-Anap fluorescence is shown in blue, immunofluorescence staining of TDP-43 in red, and the stress-granule marker G3BP1 in green (Scale bars, 10 μm).

Figure 2: Fluorescence imaging of TDP-Anap and stress granules under basal and stress conditions. Representative fluorescence images of primary mouse cortical neurons expressing TDP-Anap under untreated (NT) conditions or following sodium arsenite treatment (SA). Intrinsic TDP-Anap fluorescence is shown in blue, and immunofluorescence staining of TDP-43 is shown in red (Scale bars, 10 μm).