Minimal genetically encoded tags for fluorescent protein labeling in living neurons

Modern light microscopy, including super-resolution techniques, has brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with bioorthogonal click chemistry. The minimal size of the UAA and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision in living cells make click labeling unique. Here, we establish click labeling in living primary neurons and use it for fixed-cell, live-cell, dual-color pulse–chase, and super-resolution microscopy of neurofilament light chain (NFL). We also show that click labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced multicolor microscopy techniques, we anticipate that click labeling will contribute to novel discoveries in the neurobiology field.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. No statistical method was used to predetermine the sample size. We determined the sample size based on published work as will be described below. For the experiments involving ORANGE efficiency quantification shown in Supplementary Fig. 15e, we decided to analyze at least 500 transfected (mCherry+) neurons per timepoint. To this aim, we acquired images of all transfected neurons across three experiments. In total, 1780 images were acquired across three experiments. We analyzed 534 neurons per timepoint (178 neurons per experiment per timepoint).
In the original article describing this method (Wilems et al., 2020 https://doi.org/10.1371/journal.pbio.3000665), ORANGE efficiency was estimated by analyzing around 1000 transfected neurons from two independent neuronal cultures. We analyzed less neurons per experiment but did three experiments which overall we thought was sufficient.
For the experiments involving quantification of TKIT efficiency shown in Supplementary Fig. 17c, we decided to count 300 transfected (mCherry +) neurons per experiment. To this aim, we acquired images of all transfected neurons across two experiments. In total, 627 images were acquired. The total number of analyzed neurons was 600. In the original article describing this method (Fang et al., eLife 2021;10:e65202 doi: 10.7554/eLife.65202) TKIT efficiency was estimated by analyzing around 150-300 neurons collected across 3-4 experiments. We analyzed higher number of neurons than previous study and we considered it sufficient.
No data was excluded.
Each experiment (except for anti-FLAG immunostaining control shown in Supplementary Fig. 6c,d and TKIT incorporation efficiency shown in Supplementary Fig. 17c) was repeated at least three times. Replicated experiments were successful. Control anti-FLAG immunostaining and colocalization analysis as shown in Supplementary Fig. 6c,d was performed twice, but the same antibody was reproducibly used for anti-FLAG staining in other experiments. TKIT incorporation efficiency experiments were done twice. As described above in the section about the sample size, we analyzed significantly higher number of neurons compared to the literature (Fang et al., 2021 analyzed 150-300 neurons across 3-4 experiments and we analyzed 600 neurons across 2 experiments) and that is the reason why we did only two experiments.
Cells and neurons were randomly allocated into different experimental groups. This was done for all biological and technical replicates. To limit bias, for experiments involving comparisons of different conditions (treatments), experiments and data collection were performed in parallel. This was done for all techniques, including microscopy and western blots.
Blinding during data collection was not performed since the same investigator did the group allocation (cell/neuron seeding, transfections, click labeling etc) and the data collection. Furthermore, blinding during data collection was not feasible since the investigator had to adjust microscopy settings to fit with different fluorophore combinations. Analysis of the data presented in plots in Supplementary Figures 10 and  16c

Authentication
Mycoplasma contamination the analysis of the data presented in Supplementary Fig. 17c since it involved only one timepoint.