Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry

  1. Nevena Stajković
  2. Yuanyuan Liu
  3. Aleksandra Arsić
  4. Ning Meng
  5. Hang Lyu
  6. Nan Zhang
  7. Dirk Grimm
  8. Holger Lerche
  9. Ivana Nikić-Spiegel

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Abstract

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and click chemistry. The small size of UAAs and the possibility of introducing them virtually anywhere into target proteins make this method particularly suitable for labeling of complex and spatially restricted proteins. Using this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186; encoded by Nfasc) and the 260 kDa voltage-gated Na+ channel (NaV1.6, encoded by Scn8a) in primary neurons and performed conventional and super-resolution microscopy. We also studied the localization of epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to improve the efficiency of UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in neurons, an achievement that could be transferred to more complex systems such as organotypic slice cultures, organoids, and animal models.

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  1. Version published to 10.1242/jcs.260600
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    Reply to the reviewers

    1. General Statements

    We would like to thank the reviewers for their insightful and useful comments about our manuscript. Based on these comments and as outlined in our revision plan, we plan to strengthen our findings by performing new experiments and quantitative analyses. This particularly applies to our nanoscale (dSTORM) imaging dataset which was discussed by multiple reviewers.

    We also appreciate the reviewers’ overall positive evaluation of the significance of our labeling method for the axon initial segment studies. With regards to this, we would like to highlight that this manuscript particularly addresses the labeling of “difficult-to-label” neuronal proteins, such as large ion channels and transmembrane proteins. Although we and another group have recently reported click labeling of neurofilament light chain (PMID: 35031604) and AMPAR regulatory proteins (PMID: 34795271) in primary neurons, both of these proteins have a small size between ~30-68 kDa and compared to larger ion channels/transmembrane proteins are “easier” to express in primary neurons. The novelty in the current manuscript is that we successfully applied this method for the labeling of large and spatially restricted AIS components, such as NF186 and Nav1.6 (186 and 260 kDa, respectively). As some of the reviewers also pointed out, the size and complexity of these proteins makes labeling of the AIS rather challenging. We also used our approach to study the localization of epilepsy-causing Nav1.6 variants and could exclude the retention in the cytoplasm as a possible cause of their loss of function. Finally, we improved the efficiency of genetic code expansion in primary neurons by developing AAV-based viral vectors. Although AAVs are routinely used for gene delivery to neurons, AAVs for click-based labeling need to encode multiple components of the orthogonal translational machinery for genetic code expansion. By trying different promoters and gene combinations, we developed several variants that enable high efficiency of the genetic code expansion in neurons. On their own, these findings will facilitate further genetic code expansion and click chemistry studies, beyond the labeling of the axon initial segment.

    2. Description of the planned revisions

    Reviewer #2

    • On lines 107 and 108, the sentence "The C-terminal HA-tag allowed us to detect the full-length NF-186 protein by immunostaining it with an anti-HA antibody" would have a better place just after lines 104-105 " [...] we modified the previously described plasmid (Zhang et al., 1998) by moving the hemagglutinin (HA) tag from the N terminus to the C terminus".

    We will modify the text as the reviewer suggested.

    • Fig.2b: the AnkG staining looks substantially longer than that showed in c. However, the results on AIS length show no significant changes in between the groups. This is visually misleading, the authors should choose a picture for the WT construct that is representative of the data.

    We thank the reviewer for bringing this up. We will replace the panel in Fig.2b with a more representative image of NF186 WT construct in the revised version of the manuscript.

    • Line 238: what is the rationale behind choosing these cells? For example, have they been used in other studies for similar purposes? If so, please provide the reference.

    We initially probed neuroblastoma ND7/23 which are commonly used for the electrophysiological recordings of recombinant Nav1.6 (PMID: 30615093, 22623668, 25874799, 27375106). Although we were able to record Na+ currents in those cells, only a small portion of channels was detected on the cell surface by microscopy (Suppl. Fig. 5a). As we discuss in the manuscript (lines 237-240), we then switched to N1E-115-1 cells in which we obtained a higher level of expression of the recombinant NaV1.6 channels on the cell surface (Suppl. Fig. 5b). These cells have also been previously used for the electrophysiological studies of voltage-gated sodium channels, including Nav1.6 (PMID: 8822380, 24077057). We will modify the text and include these references in the revised manuscript.

    • Figure 3c, the authors omitted the comparison with the WT construct this time, as opposed to the neurofascin experiments. What is the reason?

    As shown by others (PMID: 31900387) and us in this manuscript, one of the main issues with the expression of the recombinant NF186 in neurons was that overexpression led to mislocalization of NF186 in neuronal soma and processes. This was particularly true for WT construct and certain amber mutants (e.g. K809TAG). Based on previous reports (PMID: 31900387), we then tested a weak human neuron-specific enolase promoter. This reduced expression level and improved localization of NF186. However, since we still observed some neurons with mislocalized NF186 WT even with the enolase promoter, we found it important to quantitively compare the AIS length of WT construct and amber mutants to surrounding untransfected cells. On the other hand, since we did not have overexpression and mislocalization problem with Nav1.6 WT construct (all observed neurons have signal localizing in the AIS), we measured only the AIS length of the amber mutants. However, to avoid any confusion, we will also measure the AIS size of the neurons expressing Nav1.6 WT construct and compare it to surrounding cells and amber mutants. For this, we will need to perform new experiments and acquire new images. We will include the data in the revised manuscript.

    • Fig. 4: why did the authors chose these cells for electrophysiology experiments and not neurons? Explain the rationale in the text or, alternatively, cite similar studies using the same tool.

    Due to the branched neuronal processes which cause the space clamp problem in voltage clamp experiments with neurons, round and none-branching cells are frequently used to examine the biophysical properties of ion channels, including Nav1.6. By far, most of studies investigating the biophysical properties of NaV1.6 channels were performed in neuroblastoma cells e.g. ND7/23 and N1E-115-1 cells (PMID: 25874799; 25242737). We tested these two types of cells and found that N1E-115-1 cells supported higher expression level of the recombinant NaV1.6 channels on the cell surface than the ND7/23 cells (Suppl. Fig 5). Hence, N1E-115-1 were more suitable to get robust and reliable recordings (as we also discuss above in the response to reviewer’s comment). We will clarify this in the revised manuscript.

    • Fig.4, biophysical properties: did the authors find differences in passive properties? Measures of resting potential, membrane resistance and cell capacitance should be reported.

    Passive properties such as resting membrane potential and membrane resistance are important functional features in neurons measured in current clamp experiments, but not applicable for ND7/23 and N1E-115-1 cells used in our voltage clamp experiments. To measure the Na+ current mediated by WT or mutant NaV1.6 channels expressed in N1E-115-1 cells, the endogenous Na+ channels were blocked by tetrodotoxin and the endogenous K+ channels were blocked by tetraethylammonium chloride, CsCl and CsF in extracellular and intracellular solutions. Under these conditions, resting potential and membrane resistance are not relevant for experiments. Cell capacitance reflects the size of the cell surface area, which can affect the number of channels expressed on the cell surface. To eliminate the effect of different cell sizes, Na+ current densities normalized by cell capacitances were used in our experiments. We will report on these values in the revised manuscript.

    • Fig 4, STORM images. The periodic distribution of the dots should be enhanced with some sort of arrows or lines, for the non-specialist audience.

    Based on the comments from multiple reviewers, we plan to obtain additional dSTORM images of the neurons expressing recombinant Nav1.6 WT or amber mutants. We also intend to improve the visualization of these results by updating/modifying existing figures and including quantitative data.

    • Line 374: rat or mouse primary neurons?

    We are here referring to both, rat and mouse neurons. The images shown in Fig. 06 and Suppl. Fig. 08 were obtained from rat cortical neurons expressing Nav1.6 or fluorescent reporter. However, we were also able to successfully transduce mouse neurons with AAV92A carrying orthogonal translational machinery (data not shown). We will clarify this in the revised manuscript.

    **Referees cross-commenting**

    I fully agree with the following remarks from Reviewers #3, #4 and #5. This is a point that I have raised in my report too. The authors need better images to show the periodicity we visualization, and a quantification would be of great benefit to support the claim with numbers (and how these compare to similar studies in the literature):

    R3: 2. For the dSTORM analysis of the tagged Nav1.6 protein, I also cannot tell there is periodic organization from the image directly. Some analysis is needed there. R4: 2."As there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), these experiments confirmed that the NaV1.6 overexpression, TCO*A319 Lys incorporation, and click labeling did not affect the nanoscale periodic organization of the sodium channels in the AIS." It is clearly noticeable that for WT, the spot density is more compared to the other two mutants. Why is that so? Using cluster analysis, one can quantify spot density and discuss nanoscale organization quantitatively. The author should quantify the periodicity and compare it among different variants and with previous reports. R5: 3. The authors claim that there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), but it is hard to conclude this without any quantification and statistical analysis. Sodium channels have been shown to be associated with the membrane-associated periodic skeleton structures in neurons and average autocorrelation analysis has been developed to quantify the degree of periodicity of such structural organizations (Han et al. PNAS 114(32)E6678-E6685, 2017). The authors should use this approach to quantify and compare the average autocorrelation amplitudes.

    As we outlined in our responses to the individual reviewers’ comments below, we will address these questions by performing new experiments and quantifications.

    I also agree with these comments from Reviewers #3 and #5:

    R3: 4. It is unclear, for all the presented data, whether all the cells are collected from a single biological replicate or from multiple replicates. At least 2-3 replicates are needed to see the reproducibility in terms of labeling efficiency, and other related conclusions. R5: 1. The authors should indicate how many replicates were performed and how many cells were analyzed for each experiment.

    We thank the reviewers for bringing this up. By mistake, we omitted this important information. We will include it in the revised manuscript, but we would like to highlight here that each experiment was repeated at least 3 times.

    Reviewer #3

    1. There is some patch-like background from the 488 channel from the click reaction, some of which have very as strong signal as the staining on the neurons. What is the potential cause for this? With immunostaining on HA, the background doesn't affect too much on the image data interpretation. However, the major goal of this method development is to use it in live-cell without immunostaining. Without another reference, the high background might cause issues in data interpretation. Can the author also suggest way to avoid or lower this in the discussion?

    We thank the reviewer for bringing this up. We have occasionally observed patch-like background in what appears to be the cell debris. Such dead cells do not have an intact cell membrane and therefore can absorb cell-impermeable ATTO488-tetrazine dye during click labeling. This kind of background is also present in the control neurons transfected with the WT Nav1.6, which suggests that it originates from the UAA and tetrazine-dye accumulations. Additionally, since these patches are not visible with the immunostaining, they do not contain our protein of interest, which further confirms that they contain only dye and UAA accumulations. Depending on the neuron prep/quality before and after transfections, the presence of these patches is more or less obvious. However, despite the background we did not have problems identifying AIS during live cell imaging. Especially when overall neuronal health is optimal after transfections, AIS can easily be distinguished from patches that are positioned outside of labeled neurons. We will investigate this further and discuss it in the revised manuscript.

    1. For the dSTORM analysis of the tagged Nav1.6 protein, I also cannot tell there is periodic organization from the image directly. Some analysis is needed there.

    We will address this in the revised manuscript by performing additional experiments and quantifications. We also wrote a detailed answer below, in the response to the other reviewers.

    1. The authors use the AIS length as a parameter to evaluate the function of the clickable mutant of NF186, and using patch clamp for functional validation of the clickable mutant of Nav1.6. In both cases, the comparison is done between the mutant and the WT construct, but both in transfected cell and exogenously expressed. It's also worth comparing with untransfected cells as the true native situation.

    We agree with the reviewer that it is important to compare transfected cells with untransfected cells. As the reviewer points out, we have already performed some of these comparisons. When it comes to the NF186, we used the AIS length as a parameter to estimate if the expression of clickable mutant affected the AIS structure. As we show in the Fig. 02, we co-immunostained neurons transfected with NF186-HA WT or TAG constructs. We used HA antibody to detect neurons expressing NF186, while the ankG was used as a marker of the AIS length. To check if the AIS length of transfected cells is affected, we compared the length of transfected cells (expressing NF186, HA+) to surrounding untransfected cells (HA-). When it comes to the Nav1.6, we also compared the AIS length of cells expressing Nav1.6 (HA+) to surrounding untransfected cells (HA-). Similarly to the experiments with NF186, this allowed us to check if the expression of the recombinant Nav1.6 affect the AIS structure. What is missing is the comparison with untransfected conditions (i.e. neurons that are simply stained with ankG). We assume that is what the reviewer is referring to? We will also include these data in the revised manuscript. Furthermore, since we introduced a labeling modification in NaV1.6, we wanted to check if such modification would affect its function. To do so, as routinely done in the field (PMID: 25874799), we rendered the WT and TAG channels TTX-resistant and recorded only recombinant Na+ currents in neuroblastoma cells in the presence of TTX. Perhaps we misunderstand the reviewer’s comment, but in this regard measurements of untransfected cells are not relevant since they would not allow us to compare WT and TAG mutants.

    1. It is unclear, for all the presented data, whether all the cells are collected from a single biological replicate or from multiple replicates. At least 2-3 replicates are needed to see the reproducibility in terms of labeling efficiency, and other related conclusions.

    We thank the reviewer for the observation. By mistake, we omitted this important information. We will include in the revised version of the manuscript. We would like to highlight here that each experiment was repeated at least 3 times.

    Reviewer #4

    1."Confocal microscopy revealed that the hNSE promoter lowered the WT and clickable NF186-HA expression levels and consequently improved the localization of these proteins." Is the lower expression level a measure of localization improvement? How does the author conclude that the localization has improved?

    Previous report (PMID: 31900387) suggested that the overexpression of the recombinant WT NF186 can affect its trafficking, leading to the NF186 mislocalization. We observed the same in our experiments with CMV NF186 (in particular for NF186 WT). Hence, based on the PMID: 31900387 we probed weak neuron specific enolase promoter. Since the WT was the most problematic in terms of the ectopic expression, we checked if AIS localization was improved with enolase promoter for this construct. To this aim, we counted number of neurons that with mislocalized signal or with the signal in the AIS for both, CMV and enolase promoter. We could observe that number of neurons with mislocalized signal was lower for enolase promoter. Since there were more neurons with the AIS-specific signal when NF186 was expressed from enolase promoter compared to CMV, we concluded that enolase promoter lowered expression and improved localization of the NF186. Therefore, we used enolase promoter for click labeling of NF186 amber mutants. We will include the results of this analysis in the revised version of the manuscript.

    2."As there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), these experiments confirmed that the NaV1.6 overexpression, TCO*A319 Lys incorporation, and click labeling did not affect the nanoscale periodic organization of the sodium channels in the AIS." It is clearly noticeable that for WT, the spot density is more compared to the other two mutants. Why is that so? Using cluster analysis, one can quantify spot density and discuss nanoscale organization quantitatively. The author should quantify the periodicity and compare it among different variants and with previous reports.

    We thank the reviewer for these suggestions. We will address these remarks by performing additional new experiments and quantifications. The difference in the level of the expression of the recombinant Nav1.6 might explain differences in the spot density for WT vs. TAG clickable mutants. However, as the reviewer suggested quantitative analysis is needed to address these concerns. We also intend to quantify the periodicity and compare it among different variants and with previous reports. It is just important to note that in the current version of the manuscript we looked at the nanoscale organization of the subset of Nav1.6 channels. The reason being that we used anti-HA antibody which will only detect our recombinant protein which got incorporated into the AIS and not the endogenous Nav1.6.

    Minor comments

    1."Although NF186K809TAG 158 -HA (Supplementary Fig. 4) showed bright click labeling, we excluded it from the analysis due to its frequent ectopic expression along the distal axon." How frequently is this bright click labeling observed for this mutation? Is it not observed for other mutations at all? The authors should state this point clearly with some statistics.

    We are not sure what is the exact question from the reviewer. If we understand it correctly, the reviewer is asking us to quantify how frequent was the ectopic expression of this amber mutant compared to other mutants? And not the click labeling (as written in their original comment), since click labeling was observed for all the mutants independently of their ectopic expression?

    2."Immunostaining with anti-HA antibody revealed that the expression of NaV1.6WT 239 -HA on the membrane of the N1E-115-1 cells was higher than on the ND7/23 cells (Supplementary Fig. 5a-c). However, click labeling of both NaV1.6K1425TAG 240 -HA and NaV1.6K1546TAG 241 -HA with ATTO488-tz was not successful (Supplementary fig. 5d) indicating insufficient expression of the clickable constructs." Is this due to insufficient expression level or accessibility? The author should make this statement clear.

    We thank the reviewer for bringing this up. We will clarify this in the revised version of the manuscript. We believe that the click labeling of the K1546TAG mutant in N1E-115-1 cells is absent due to the insufficient expression of the channels on the membrane, since this mutant was successfully labeled in the primary neurons that represent more native environment and where Nav1.6 form high-density clusters. K1425TAG mutant is not labeled due to the insufficient expression on the membrane in N1E-115-1 cells as well. However, since this mutant is also poorly labeled in primary neurons, we can speculate that K1425TAG position might be less accessible for the tetrazine-dye compared to K1546TAG. To further support our claim that due to the insufficient expression click labeling is low/absent in neuronal cells, we can use NF186 as an additional example. When NF186 was expressed from strong CMV promoter, we observed click labeling for all the mutants in ND7/23 cells (Suppl. Fig.01). However, when CMV was replaced with neuron specific enolase promoter, the expression was of NF186 was substantially lower in ND7/23 cells and click labeling was absent (data not shown). We will clarify this in the revised manuscript.

    1. Authors should clearly state the drift correction procedure of 3D STORM data. What are the localization precision and photon count for 3D STORM experiments?

    We processed 3D dSTORM data in NIS-elements AR software. We used the automatic drift correction from the NIS-elements software that is based on the autocorrelation. We will provide further and updated information in the revised manuscript, including the localization precision and photon count for the new dSTORM images.

    1. "Click labeling of NaV1.6 channels in living primary neurons" What kind of primary neurons have been used for click labeling of NaV1.6 channels? Is there any specific reason why authors have chosen cortical neurons for labeling NF186? Does this labeling strategy depend on primary neuron type?

    For the establishment and click labeling of Nav1.6 we used primary rat cortical neurons (Fig. 03, Fig. 06). The same neuronal type has been used for click labeling of NF186 (Fig. 02). We established labeling of the AIS components in cortical neurons because we use those routinely in the laboratory. However, this labeling strategy does not depend on the neuronal type. As we show in Fig. 05, to study localization of the loss-of-function pathogenic Nav1.6 variants we used mouse hippocampal neurons. The reason for this is that in previous study the same neuronal type was used to characterize these two mutations (lines 361-362). This demonstrates nicely that method can be easily transferred to any neuronal type. Furthermore, we were also able to label Nav1.6 and NF186 in mouse cortical neurons (data are not shown in the manuscript). We will clarify this in the revised manuscript.

    Reviewer #5

    1.Throughout the manuscript, only one representative image containing one AIG is shown for each condition without statistics and quantifications, so the conclusions are not sufficiently convincing. For example, in Fig. 1b, c, e; Fig. 2b,c,d,e; Fig. 3b,c,d,e ; Fig. 5c; and Supplementary Fig.1-6, the authors should quantify the average fluorescence intensities both for HA immunostaining and ATTO488-tz labeling in different conditions, as well as the labeling ratios (fluorescence intensity ratios between ATTO488 and AF647/AF555) . Without statistics and quantifications, it is unclear whether there is any significant difference between the constructs with different TAG positions, or between different transfection methods (e.g., lipofectamine 2000 vs 3000).

    We agree with the reviewer that the quantitative analysis is important and we will provide more quantitative data in the revised manuscript. At the same time, we are a bit confused by this comment which seems to refer to missing quantifications in one of the schemes (Fig. 1) and overlooks existing quantifications (e.g. quantitative analysis of the data set from Fig. 5c is shown in Fig. 5d). However, as suggested by the reviewer and to strengthen our data, in addition to the quantifications already provided in the manuscript (e.g. Fig. 2d: AIS length of NF186TAG constructs; Fig. 3f: AIS length of Nav1.6 TAG constructs; Fig. 5d: click-labeling intensity of LOF mutants), we intend to quantify the differences between labeling ratios of different mutants and transfection methods. When it comes to the different transfection methods, some data is already provided in the manuscript (e.g. we counted number of transfected versus transduced neurons) but we intend to clarify and expand on this in the revised manuscript.

    1. The only quantification done was for the average AIS length, but the statistical tests should be performed between different conditions and the corresponding P values should be provided. It seems that the transfected neurons generally have a longer AIS length than the transfected neurons (Fig. 2d and 3f). Could the authors provide an explanation for this?

    We are a bit confused by the first part of this comment. We measured the AIS lengths of NF186 WT or NF186 TAG as well as Nav1.6 TAG and compared it to the AIS lengths of surrounding untransfected cells (Fig. 2d and Fig.03f). In addition, we compared the AIS lengths of the NF186 WT and TAG to each other, and Nav1.6 TAG to each other. To analyze the differences, we performed statistical tests and provided the corresponding p values in the figure legends (Fig. 02 and 03). Further details on the statistical analysis are provided in supplementary tables (Suppl. table 01 and 02). Regarding the 2nd question, we have also noticed that the AIS lengths of transfected neurons appear longer than those of untransfected cells. This seems to be more pronounced in the case of NF186 which is expressed at the higher level compared to the Nav1.6. The appearance of slightly longer AIS is most likely the consequence of the fact that recombinant constructs are overexpressed in the neurons that express endogenous NF186 and Nav1.6. However, this difference in the AIS length is not significant to the controls. We will discuss this further in the revised manuscript.

    1. The authors claim that there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), but it is hard to conclude this without any quantification and statistical analysis. Sodium channels have been shown to be associated with the membrane-associated periodic skeleton structures in neurons and average autocorrelation analysis has been developed to quantify the degree of periodicity of such structural organizations (Han et al. PNAS 114(32)E6678-E6685, 2017). The authors should use this approach to quantify and compare the average autocorrelation amplitudes.

    We are thankful to the reviewer for suggestions on how to quantify the periodicity of recombinant sodium channels and how to more accurately compare WT and TAG mutants at the nanoscale level. We will perform additional experiments and analysis in order to address the concerns of this and other reviewers.

    1. The authors should also obtain dSTORM images for the click labeled neurons to demonstrate if the click labeling method would provide sufficient labeling efficiency for dSTORM, compared to immunostaining (HA and Ankyrin G immunostaining).

    We would like to thank the reviewer for this suggestion. We have already shown in our previous work that STED can be performed with click labeled neurons (PMID: 35031604). When it comes to this manuscript and AIS labeling, we have already obtained preliminary dSTORM images of click-labeled NF186. Since the expression of Nav1.6 is lower compared to NF186, the labeling is also less bright and dSTORM is a bit more challenging. To try to overcome this issue, in addition to dSTORM of click-labeled Nav1.6, we are planning to try click-PAINT (PMID: 27804198). Click-PAINT has been used for super-resolution imaging of less abundant targets in cells and could possibly allow super-resolution imaging of Nav1.6. We will report on these new experiments in the revised version of the manuscript.

    1. It seems that the click labeling has a off-target/background labeling in the soma of the neuron (see Fig. 3c,d. Could the authors quantify and determine the sources of such off-target labeling?

    We thank the reviewer for pointing this out. We will clarify this in the revised manuscript, but by looking at the other examples from our dataset it appears to us that this background is present in WT constructs as well. In the current version of the manuscript, this is not clear since the WT image that is shown in the Fig. 03b is a single plane confocal image. Therefore, we will replace it in the revised manuscript with a z-stack in which the presence of the background is more obvious (due to the maximum intensity projection). In addition, we will conduct additional control experiments to clarify this.

    Minor comments:

    1. The authors should indicate how many replicates were performed and how many cells were analyzed for each experiment.

    We thank the reviewer for bringing this up. By mistake, we omitted this important information. We will include this information in the revised manuscript, but we would like to highlight here that each experiment was repeated at least 3 times.

    1. The display range (i.e., intensity scale bar) was indicated only for a small portion of the fluorescence images. It is better to be consistent and show the display range for all images presented.

    We will include intensity scale bars in all the images in the revised version of the manuscript.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Not applicable.

    4. Description of analyses that authors prefer not to carry out

    Reviewer #3, comment #5. One application presented in this manuscript is to evaluate the effect of epilepsy-causing mutations of Nav1.6. By comparing the intensity of ATTO488, the result suggests that there is no significant impact of these mutations on membrane tracking. I am wondering if the author should study the membrane tracking by also looking at the diffusion in live-cell with the labeling method. The comparison of the intensity only can be achieved by just immunostaining. It doesn't really demonstrate the benefit of live-cell labeling and imaging with the presented method.

    Generally speaking, one of the advantages of click labeling is its compatibility with live cell labeling. As the reviewer also points out, this is especially useful for live-cell imaging but is not limited to it. In addition, click labeling allows selective labeling of membrane population of Nav1.6 in living neurons. We took advantage of this and used cell-impermeable dyes to label unnatural amino acids incorporated into extracellular part of Nav1.6 (Scheme 03a). On the contrary, HA tag that allows immunodetection of recombinant Nav1.6 is added to the intracellular C terminus. Hence, by anti-HA immunostaining total (intra- and extracellular) epilepsy-causing Nav1.6 channel population will be detected. That is why in this case live-cell click labeling was advantageous compared to the conventional immunostaining. We will clarify this in the revised manuscript. In addition, we would like to note that when we started the experiments with the epilepsy-causing mutations, we wanted to a) check if they are present on the membrane and b) depending on the outcome of those experiments follow the trafficking of these LOF Nav1.6 mutants. Since patch clamp recordings of pathogenic Nav1.6 showed loss of Na+ currents, we at first assumed that they are not properly expressed on the membrane. However, our click labeling showed that the pathogenic channels were detected at the AIS membrane despite the loss of Na+ currents. This was also somewhat surprising to us and we would love to investigate this further. We also appreciate the reviewer’s suggestion in this regard and we hope to be able to use all the advantages of our labeling approach in our follow-up studies. However, keeping in mind time and resources limitations, live-cell trafficking study might be beyond the scope of this revision.

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    Referee #5

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Nevena Stajković et al. present a method for live labeling of the proteins localized at the axon initial segment (AIS) of cultured neurons using unnatural amino acids (UAAs) carrying strained alkenes and click chemistry. Using this method, the authors showed the successful labeling of two AIS-localized proteins, the 186 kDa isoform of neurofascin (NF186) and the 260 kDa voltage-gated sodium channel (NaV1.6). The authors also showed the transduction of neurons using adeno-associated viruses (AAVs) had higher efficiency than transfection by lipofectamine in delivering the vectors expressing required components for the click labeling.

    Major comments:

    1. Throughout the manuscript, only one representative image containing one AIG is shown for each condition without statistics and quantifications, so the conclusions are not sufficiently convincing. For example, in Fig. 1b, c, e; Fig. 2b,c,d,e; Fig. 3b,c,d,e ; Fig. 5c; and Supplementary Fig.1-6, the authors should quantify the average fluorescence intensities both for HA immunostaining and ATTO488-tz labeling in different conditions, as well as the labeling ratios (fluorescence intensity ratios between ATTO488 and AF647/AF555) . Without statistics and quantifications, it is unclear whether there is any significant difference between the constructs with different TAG positions, or between different transfection methods (e.g., lipofectamine 2000 vs 3000).
    2. The only quantification done was for the average AIS length, but the statistical tests should be preformed between different conditions and the corresponding P values should be provided. It seems that the transfected neurons generally have a longer AIS length than the transfected neurons (Fig. 2d and 3f). Could the authors provide an explanation for this?
    3. The authors claim that there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), but it is hard to conclude this without any quantification and statistical analysis. Sodium channels have been shown to be associated with the membrane-associated periodic skeleton structures in neurons and average autocorrelation analysis has been developed to quantify the degree of periodicity of such structural organizations (Han et al. PNAS 114(32)E6678-E6685, 2017). The authors should use this approach to quantify and compare the average autocorrelation amplitudes.
    4. The authors should also obtain dSTORM images for the click labeled neurons to demonstrate if the click labeling method would provide sufficient labeling efficiency for dSTORM, compared to immunostaining (HA and Ankyrin G immunostaining).
    5. It seems that the click labeling has a off-target/background labeling in the soma of the neuron ( see Fig. 3c,d. Could the authors quantify and determine the sources of such off-target labeling?

    Minor comments:

    1. The authors should indicate how many replicates were performed and how many cells were analyzed for each experiment.
    2. The display range (i.e., intensity scale bar) was indicated only for a small portion of the fluorescence images. It is better to be consistent and show the display range for all images presented.

    Significance

    Unnatural amino acid (UAA)-based minimal tags for live-cell protein labeling in mammalian cells were invented about ten years ago (Lang et al., 2012b, Lang et al., 2012a, Nikic et al., 2014, Plass et al., 2012, Uttamapinant et al., 2015), and these authors recently introduced this labeling method to label live cultured neurons (Arsić et al., 2022). Therefore, it is unclear whether the method present in this manuscript has any significant advance compared to the Arsić et al. paper, given that the major difference between the two papers is that in the current manuscript, AIS localized proteins were labeled, whereas in the Arsić et al. paper, neurofilaments were labeled in the neurons. Therefore, the method presented in the current manuscript does not provide much novelty or technical advance compared to what has been described in the Arsić et al. paper.

    My expertis is super-resolution flurescence imaging, cell labeling methods, and neurobiology.

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    Referee #4

    Evidence, reproducibility and clarity

    The manuscript demonstrates a novel method of labeling two large components of the initial axon segment, neurofascin (NF186) and Nav1.6 using unnatural amino acids and click chemistry in live cells. They have applied their method for epilepsy causing two Nav1.6 variants without affecting their functionality. Since these proteins are larger in size, selecting the labeling sites and transfection efficiency become critical factors. They have targeted different lysine sites and shown the best performing labeling site. Also, they have developed a viral vector to improve transfection efficiency.

    The experiments are well designed, and the manuscript is nicely written. In my opinion, the manuscript can be accepted, but the author should address the following comments.

    Major comments

    1. "Confocal microscopy revealed that the hNSE promoter lowered the WT and clickable NF186-HA expression levels and consequently improved the localization of these proteins." Is the lower expression level a measure of localization improvement? How does the author conclude that the localization has improved?
    2. "As there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), these experiments confirmed that the NaV1.6 overexpression, TCO*A319 Lys incorporation, and click labeling did not affect the nanoscale periodic organization of the sodium channels in the AIS." It is clearly noticeable that for WT, the spot density is more compared to the other two mutants. Why is that so? Using cluster analysis, one can quantify spot density and discuss nanoscale organization quantitatively. The author should quantify the periodicity and compare it among different variants and with previous reports.

    Minor comments

    1. "Although NF186K809TAG 158 -HA (Supplementary Fig. 4) showed bright click labeling, we excluded it from the analysis due to its frequent ectopic expression along the distal axon." How frequently is this bright click labeling observed for this mutation? Is it not observed for other mutations at all? The authors should state this point clearly with some statistics.
    2. "Immunostaining with anti-HA antibody revealed that the expression of NaV1.6WT 239 -HA on the membrane of the N1E-115-1 cells was higher than on the ND7/23 cells (Supplementary Fig. 5a-c). However, click labeling of both NaV1.6K1425TAG 240 -HA and NaV1.6K1546TAG 241 -HA with ATTO488-tz was not successful (Supplementary fig. 5d) indicating insufficient expression of the clickable constructs." Is this due to insufficient expression level or accessibility? The author should make this statement clear.
    3. Authors should clearly state the drift correction procedure of 3D STORM data. What are the localization precision and photon count for 3D STORM experiments?
    4. "Click labeling of NaV1.6 channels in living primary neurons" What kind of primary neurons have been used for click labeling of NaV1.6 channels? Is there any specific reason why authors have chosen cortical neurons for labeling NF186? Does this labeling strategy depend on primary neuron type?

    Significance

    Although the use of unnatural amino acids and click chemistry for labelling has been shown before from the same group, labelling large proteins, especially ion channels, without affecting their function is always challenging because of the accessibility of the labelling site as well as poor transfection efficiency. Here, they have selected two such large essential proteins: NF186 and Nav1.6, which are associated with epilepsy, and developed a method for fluorophore labelling with minimal perturbation. Other approaches namely using fluorescent proteins, biotin-streptavidin chemistry and halo-tag have been reported before to label these proteins, but these have a strong impact on their mislocalisation and perturbing their functionality. Therefore, this method will be of great importance in the field of studying these proteins.

    Expertise: Live-cell confocal and multi-photon microscopy imaging, Super-resolution microscopy imaging, Live-cell labelling, and Amyloid aggregations

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Stajkovic et al the describes step-wise generation and validation of the fluorescent labeling of NF186 and Nav1.6 in primary neurons by non-natural amino acid and click chemistry. For each protein of interest, the authors started by generating constructs carrying amber codon at different positions, and then selected for the best construct(s) by judging (1) the labeling efficiency, (2) whether the particular labeling position affect the function of the protein, and (3) whether the labeled protein shows any mislocalization. During the trouble shooting process, the authors also introduced adeno-associated viral (AAV) vectors for more efficiently delivering constructs into the cells. The method described in the manuscript could become a reference for researchers who aim to label similar neuronal proteins.

    Specific comments:

    1. There is some patch-like background from the 488 channel from the click reaction, some of which have very as strong signal as the staining on the neurons. What is the potential cause for this? With immunostaining on HA, the background doesn't affect too much on the image data interpretation. However, the major goal of this method development is to use it in live-cell without immunostaining. Without another reference, the high background might cause issues in data interpretation. Can the author also suggest way to avoid or lower this in the discussion?
    2. For the dSTORM analysis of the tagged Nav1.6 protein, I also cannot tell there is periodic organization from the image directly. Some analysis is needed there.
    3. The authors use the AIS length as a parameter to evaluate the function of the clickable mutant of NF186, and using patch clamp for functional validation of the clickable mutant of Nav1.6. In both cases, the comparison is done between the mutant and the WT construct, but both in transfected cell and exogenously expressed. It's also worth comparing with untransfected cells as the true native situation.
    4. It is unclear, for all the presented data, whether all the cells are collected from a single biological replicate or from multiple replicates. At least 2-3 replicates are needed to see the reproducibility in terms of labeling efficiency, and other related conclusions.
    5. One application presented in this manuscript is to evaluate the effect of epilepsy-causing mutations of Nav1.6. By comparing the intensity of ATTO488, the result suggests that there is no significant impact of these mutations on membrane tracking. I am wondering if the author should study the membrane tracking by also looking at the diffusion in live-cell with the labeling method. The comparison of the intensity only can be achieved by just immunostaining. It doesn't really demonstrate the benefit of live-cell labeling and imaging with the presented method.

    Significance

    The data itself is mostly convincing, however, I do not see much novelty from this manuscript. Both the labeling method using non-natural amino acid and click chemistry and AAV delivery are established. However, I can see that for research groups who specifically interested in studying these two proteins or proteins closed related, the results from this manuscript could be of direct help.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    This study proposes a novel tool for AIS live and fixed labelling based on biorthogonal click chemistry. Stajkovic and colleagues used this method to specifically label the AIS proteins NF186 and Nav1.6 of mouse and rat neurons, and did a thorough process of optimization to get convincing results. The authors considered different promoters, transfection strategies and the use of AAVs to get the most efficient labelling strategy for both proteins. They have also gone through a strong validation process based on transfection efficiency, quality of staining, potential effects on AIS length and nanostructure, and electrophysiological properties. Finally, Stajkovic and colleagues used this tool to study how two epilepsy-causing Nav1.6 mutant variants affect AIS function, providing interesting data to the understanding of this pathology. In summary, this method convincingly overcomes some well-described issues associated with pre-existing AIS live cell labelling tools by being minimally "invasive" to the proteins of interest. Besides the scientific content, another strong point of this article is the clarity of the manuscript and the figures: the presence of schematics (i.e. Fig. 1) and the detailed description of experiments and results will help non-specialist readers to follow the study. I strongly recommend this article for journal publication.

    Major comments:

    I have no major comments

    Minor comments:

    I have some minor comments:

    • On lines 107 and 108, the sentence "The C-terminal HA-tag allowed us to detect the full-length NF-186 protein by immunostaining it with an anti-HA antibody" would have a better place just after lines 104-105 " [...] we modified the previously described plasmid (Zhang et al., 1998) by moving the hemagglutinin (HA) tag from the N terminus to the C terminus".
    • Fig.2b: the AnkG staining looks substantially longer than that showed in c. However, the results on AIS length show no significant changes in between the groups. This is visually misleading, the authors should choose a picture for the WT construct that is representative of the data.
    • Line 238: what is the rationale behind choosing these cells? For example, have they been used in other studies for similar purposes? If so, please provide the reference.
    • Figure 3c, the authors omitted the comparison with the WT construct this time, as opposed to the neurofascin experiments. What is the reason?
    • Fig. 4: why did the authors chose these cells for electrophysiology experiments and not neurons? Explain the rationale in the text or, alternatively, cite similar studies using the same tool.
    • Fig.4, biophysical properties: did the authors find differences in passive properties? Measures of resting potential, membrane resistance and cell capacitance should be reported.
    • Fig 4, STORM images. The periodic distribution of the dots should be enhanced with some sort of arrows or lines, for the non-specialist audience.
    • Line 374: rat or mouse primary neurons?

    Referees cross-commenting

    I fully agree with the following remarks from Reviewers #3, #4 and #5. This is a point that I have raised in my report too. The authors need better images to show the periodicity visualization, and a quantification would be of great benefit to support the claim with numbers (and how these compare to similar studies in the literature):

    R3: 2. For the dSTORM analysis of the tagged Nav1.6 protein, I also cannot tell there is periodic organization from the image directly. Some analysis is needed there. R4: 2."As there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), these experiments confirmed that the NaV1.6 overexpression, TCO*A319 Lys incorporation, and click labeling did not affect the nanoscale periodic organization of the sodium channels in the AIS." It is clearly noticeable that for WT, the spot density is more compared to the other two mutants. Why is that so? Using cluster analysis, one can quantify spot density and discuss nanoscale organization quantitatively. The author should quantify the periodicity and compare it among different variants and with previous reports. R5: 3. The authors claim that there was no obvious difference in the nanoscale organization of the NaV1.6WT 317 -HA or NaV1.6TAG 318 -HA channels (Fig. 4. e-g), but it is hard to conclude this without any quantification and statistical analysis. Sodium channels have been shown to be associated with the membrane-associated periodic skeleton structures in neurons and average autocorrelation analysis has been developed to quantify the degree of periodicity of such structural organizations (Han et al. PNAS 114(32)E6678-E6685, 2017). The authors should use this approach to quantify and compare the average autocorrelation amplitudes.

    I also agree with these comments from Reviewers #3 and #5:

    R3: 4. It is unclear, for all the presented data, whether all the cells are collected from a single biological replicate or from multiple replicates. At least 2-3 replicates are needed to see the reproducibility in terms of labeling efficiency, and other related conclusions. R5: 1. The authors should indicate how many replicates were performed and how many cells were analyzed for each experiment.

    Significance

    The proposed tool in this article represents a big step forward in the field of AIS live cell imaging. As stated by the authors in the introduction, previous studies have described methods based on tagging fluorescent proteins to the protein of interest or labelling the extracellular part of proteins with antibodies. The same studies reported several issues: the interference with important domains of the protein due to the size and the position of the tag in the case of fluorescent proteins (Dumitrescu et al., 2016, PMID: 27932952; Dzhashiashvili et al., 2007, PMID: 17548513), or the failure to report plasticity changes in the AIS in the case of antibodies (Dumitrescu et al., 2016, PMID: 27932952). This tool can be useful for research teams aiming to understand, for example, the live development of the AIS or understanding the trafficking of its proteins. The authors have applied this method to two transmembrane proteins (NF186 and Nav1.6), but as they state in their discussion, it will be useful to tag other candidates, including cytoplasmic proteins. One of the main problems of immunocytochemistry is to find the right antibody to detect your protein. Sometimes, absence of proof is not proof of absence: just because the protein is not detected via immunostaining does not mean that the protein is not expressed there. This tool offers an alternative to these challenging scenarios.

    My expertise keywords: axon initial segment, neuronal polarity, axon biology, super resolution microscopy.

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    Referee #1

    Evidence, reproducibility and clarity

    AIS organization is highly complex and unique and AIS labeling in living cells has been problematic. In this study, authors comprehensively searched and optimized live cell markers for AIS.The main advantage of the labeling approach is that the small fluorescent dye is directly attached to the proteins of interest. Therefore, the protein of interest is modified in a minimally invasive way.

    Significance

    I was first wondering whether tool development is enough important to be published as such but here, the development and testing of the constructs, whether they affect cell functionality, is very comprehensive. This all makes this tool very useful for other researchers. I can take the method directly to use and I don't need to do all testing by myself. I feel that this tool development takes field forward.

    Own expertise: I have personally gone through the AIS live cell labeling problems and therefore I can easily appreciate the work done here.

  8. Version published to 10.1101/2022.03.01.480798v1 on bioRxiv