Interaction between Teneurin-2 and microtubules via EB proteins provides a platform for GABAA receptor exocytosis

  1. Sotaro Ichinose
  2. Yoshihiro Susuki
  3. Nobutake Hosoi
  4. Ryosuke Kaneko
  5. Mizuho Ebihara
  6. Hirokazu Hirai
  7. Hirohide Iwasaki

  • Curated by eLife

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    This potentially important paper investigates the mechanisms that contribute to building inhibitory synapses through differential protein release from microtubules. The experiments are generally designed well, but the evidence supporting the conclusions is incomplete. This manuscript will be of interest to neuroscientists and cell biologists interested in intracellular trafficking and synapse maturation.

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Abstract

Neurons form dense neural circuits by connecting to each other via synapses and exchange information through synaptic receptors to sustain brain activities. Excitatory postsynapses form and mature on spines composed predominantly of actin, while inhibitory synapses are formed directly on the shafts of dendrites where both actin and microtubules (MTs) are present. Thus, it is the accumulation of specific proteins that characterizes inhibitory synapses. In this study, we explored the mechanisms that enable efficient protein accumulation at inhibitory postsynapse. We found that some inhibitory synapses function to recruit the plus end of MTs. One of the synaptic organizers, Teneurin-2 (TEN2), tends to localize to such MT-rich synapses and recruits MTs to inhibitory postsynapses via interaction with MT plus-end tracking proteins EBs. This recruitment mechanism provides a platform for the exocytosis of GABA A receptors. These regulatory mechanisms could lead to a better understanding of the pathogenesis of disorders such as schizophrenia and autism, which are caused by excitatory/inhibitory (E/I) imbalances during synaptogenesis.

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    Author Response

    Reviewer #1 (Public Review):

    Ichinose et al., utilize a mixture of cultured hippocampal neurons and non-neuronal cells to identify the role of the transmembrane protein teneurin-2 (TEN-2) in the formation of inhibitory synapses along the dendritic shaft. First, they identify distinct clusters of gephyrin that are either actin-rich, microtubule-rich or contain neither actin nor microtubules and find that TEN-2 is enriched in microtubule-rich gephyrin clusters. This leads the authors to hypothesize that TEN-2 recruits microtubules (MTs) through the plus end binding protein EB1 when successfully matched with a pre-synaptic partner, and perform a variety of experiments to test this hypothesis. The authors then extend this finding to state quite strongly throughout the paper, including in the title, that TEN-2 acts as a signpost for the unloading of cargo from motor proteins without providing any supporting evidence. They use previous work to justify this conclusion, but without actual experiments to back up the claim, it seems like a reach.

    The strength of the paper lies in the various lines of evidence that the authors employ to assess the role of TEN-2 in MT recruitment and synaptogenesis. They have also been very thorough in validating the expression and functionality of various knock-in constructs, knock-down vectors and antibodies that were generated during the study. However, there are some discrepancies in the findings that have not been addressed satisfactorily, as well as some instances where the data presented is not of sufficient quality to support the conclusions derived from them.

    Firstly, we would like to express our sincere appreciation to Reviewer #1 for providing valuable feedback. We have carefully considered Reviewer #1 suggestions and have made significant improvements to the manuscript in response. Additionally, we have conducted an additional experiment to address the missing aspects identified in the initial submission. Furthermore, we have refined the focus of our investigation by narrowing down the number of aspects we aimed to prove and instead increased the number of confirmatory experiments. Specifically, we decided to give up on two aspects: the relationship between kinesins and cargo, and the immobilization of TEN2 in synapses (i.e., extracellular binding of TEN2). Instead, we focused on emphasizing the role of TEN2 as a platform for exocytosis. These modifications have significantly enhanced the quality of our research.

    1. The emphasis placed on the clustering analysis presented in figure 1 and the two associated supplementary figures is puzzling, since the conclusion derived from the results presented would be that Neuroligin 2 (NLGN2) is the strongest candidate to test for a relationship to MT recruitment at inhibitory post synapses. Instead, the authors cite prior evidence to exclude NLGN2 from subsequent analysis and choose to focus on TEN2 instead.

    We fully agree on the importance of studying NLGN2, as highlighted in the DISCUSSION section (line 463-471). While the cluster analysis suggests a correlation between NLGN2 and microtubules, previous research has reported microtubule localization outside the NLGN2 region (Uchigashima et al., 2016). However, this interpretation is based on EM observations at a single time point, so it will be important to evaluate it over time. Conversely, we had believed that further investigations are needed to explore the potential interactions between TEN2 and microtubules, because of its relatively limited characterization (line 156-161).

    1. It is difficult to reach the same conclusion as the authors from the images and intensity plot shown on Figure 2 E and F. While there seems to be an obvious reduction in expression levels between the TEN2N-L and TEN2TM constructs, neither seem to co-localize with EB1.

    As Reviewer #1 pointed out, the previous plots on Figure 2 were of very poor quality. Due to the dynamics of microtubules, evaluating interactions using fixed cells has limitations. Therefore, we decided to shift to live-imaging. Firstly, we observed a tendency for EB3 comets to pause at inhibitory postsynapses (Figures 1D-H). This suggests the presence of a microtubule recruiter at inhibitory synapses. Next, in dendrites expressing TEN2N-L, the velocity of EB3 comets was significantly faster compared to dendrites expressing TEN2TM or TEN2N-L2mut (Figures 7A-E). This suggests that the dominant-negative effect of TEN2N-L inhibits the function of endogenous microtubule recruiters. Additionally, the interaction between TEN2 and EB1/3 has been confirmed by GST pull-down (Figure 6A). Based on these reasoning, we propose that TEN2 present in inhibitory synapses plays a role as a microtubule recruiter through its interaction with EB1/3.

    1. The authors mimic the activity of TEN-2 at the inhibitory post synapse in non-neuronal cells by immobilizing HA- tagged TEN constructs in COS-7 cells as a proxy for synaptic partner matching. Using this model, they find that by immobilizing TEN2N-L, which contains EB1 binding motifs, MTs are excluded from the cell periphery (Figure 3D). This contradicts their conclusion that MTs are recruited through EB1 by TEN-2 on synaptic partner matching. Later in the paper, when they use the same TEN2N-L construct as a dominant negative in neuronal cells, they find that MTs are recruited the membrane, even if TEN2N-L is not immobilized by synaptic partner matching (Figure 6C). Taken together, these findings call into question the sequence of events driven by TEN-2 during synaptogenesis.

    We believe that the differences in the results between the COS-7 and neuron experiments are influenced by variations in the intracellular protein composition and distribution between COS-7 cells and neurons. Therefore, we consider it inappropriate to directly apply the results from COS-7 to neurons. Additionally, we attempted to replicate the experiments in neurons; however, it is worth mentioning that the culture of neurons on antibodies led to a significant decrease in cell viability. As a result, we have decided to withdraw the experiment of immobilized TEN2 using antibodies.

    1. It is unclear how the authors could conclude that TEN-2 is at the semi-periphery (?) of inhibitory post synapses from the STORM data that is presented in the paper. Figure 4D and 4F show comparisons of Bassoon and TEN-2 localization vs TEN-2 and gephyrin, but the image quality is not sufficient to adequately portray a strong distinction in the distance of center of mass, which is also only depicted for the TEN2-Gephyrin pair and not the TEN2-Bassoon pair in Figure 4J.

    The quality limitations of attempting a three-color dSTORM of TEN2-bassoon-gephyrin were addressed by modifying it to a two-color dSTORM. To confirm this modification, a two-color STORM was performed using VGAT instead of Bassoon (Figure 3E). The statement that TEN2 localizes to half of the synapse is supported by the observation of TEN2-gephyrin in the postsynaptic area. This observation aligns with the localization of microtubules at the postsynapse as observed by electron microscopy (Gulley & Reese, 1981; Linsalata et al., 2014).

    1. The authors do not satisfactorily explain why gephyrin appears to have completely disappeared in the TEN2N-L condition (Figure 6A), instead of appearing uniformly distributed as one would expect if MTs are indiscriminately recruited to the membrane by the dominant negative construct that remains unanchored.

    As pointed out by Reviewer #1, it needed to be adequately proven, and we mistakenly conflated dominant-negative and gain-of-function effects. However, through the examination of live imaging of EB3, observation of the localization of gephyrin, and the additional investigation of GABAAR localization in neurons expressing partial domains of TEN2, we found that TEN2N-L functions as a dominant-negative, inhibiting the microtubule recruitment function of endogenous TEN2 (Figure 7). On the other hand, it does not exhibit a gain-of-function effect in inducing exocytosis of GABAAR because both gephyrin and GABAAR were found to be reduced in the neurons expressing TEN2N-L (Figure 7F-H). Therefore, we have corrected this point.

    1. In a similar critique to that of Figure 2E and F, the distinction that the authors wish to portray between the effect of TEN2TM and TEN2N-L constructs on EGFP-TEN-2 and MAP2 colocalization (Figure 6 E and F) appear to be driven by a difference in overall expression levels of EGFP-TEN2 rather that a true difference in localization of TEN-2 and MTs.

    Regarding the previous co-localization of TEN2 and microtubules after permeabilization with saponin, we have removed it from the analysis because it is not possible to perform accurate quantitative analysis in this case. We speculate that this is a combination of two factors: the variation in transfection efficiency and the inherent variability in permeabilization between neurons. Specifically, it is particularly challenging to standardize and quantify the variability in permeabilization. Instead, the current version proposes TEN2-MT interaction via EBs by live imaging of EB3 in neurons expressing each partial domain. As observed in COS-7 cells where EB was overexpressed, whether TEN2 engages in continuous binding with microtubules or if it is a transient interaction remains an interesting topic for future investigation. We have mentioned this in the DISCUSSION section as well (line 415-422).

    Reviewer #2 (Public Review):

    Maturation of inhibitory synapses requires multiple vital biological steps including, i) translocation of cargos containing GABAARs and scaffolds (e.g. gephyrin) through microtubules (MTs), ii) exocytosis of inhibitory synapse proteins from cargo followed by the incorporation to the plasma membrane for lateral diffusion, and iii) incorporation of proteins to inhibitory synaptic sites where gephyrin and GABAARs are associated with actin. A number of studies have elucidated the molecular mechanisms for GABAARs and gephyrin translocation in each step. However, the molecular mechanisms underlying the transition between steps, particularly from exocytosis to lateral diffusion of inhibitory proteins, still need to be elucidated. This manuscript successfully characterizes three stages of inhibitory synapses during maturation, cluster1: an initial stage that receptors are being brought in and out by the MT system; cluster2: lateral diffusion stage; cluster 3: matured postsynapses anchored by gephyrin and actin, by quantifying the abundance of MAP2 or Actin in inhibitory synapse labeled by gephyrin. Importantly, the authors' findings suggest that TEN2, a trans-synaptic adhesion molecule that has two EB1 binding motifs, plays an important role in the transition from clusters 1 to 2, and inhibitory synapse maturation. The imaging results are impressive and compelling, these data will provide new insights into the mechanisms of protein transport during synapse development. However, the present study contains several loose ends preventing convincing conclusions. Most importantly, (1) it remains more TEN2 domain characterization on inhibitory synapse maturation, (2) further validation of the HA knock-in TEN2 mouse model is required, and (3) it requires additional physiology data that complement the authors' findings.

    First we would like to thank Reviewer #2 very much for the efforts and numerous suggestions. While it is highly appealing to comprehensively explain the function of a single synapse organizer in a step-by-step manner during synapse formation, we believe that it requires the identification of changing binding partners at each step, which is currently a challenging task. Therefore, in this paper, we have focused solely on the interaction between TEN2 and microtubules. As a result, we have discovered that TEN2 provides a platform for the exocytosis of GABAR, and this process relies on the interaction between TEN2 and microtubules. The analysis of the immobilization of TEN2, which was included in the previous version, will be part of a future publication. We also plan to continue detailed analysis of other domains. Thus, issues remain regarding the analysis of TEN2, but in order to confirm what is happening in just specific one step, we have made significant revisions in this revised manuscript, including analysis in HA knock-in neurons and electrophysiological analysis. We would greatly appreciate it if Reviewer #2 would reconsider the revised manuscript.

    Reviewer #3 (Public Review):

    In this paper, Ichinose et al. examine mechanisms that contribute to building inhibitory synapses through differential protein release from microtubules. They find that tenurin-2 plays a role in this process in cultured hippocampal neurons via EB1 using a variety of genetic and imaging methods. Overall, the experiments are generally designed well, but it is unclear whether their findings offer a significant advance. The experimental logic flow and rational difficult for readers to follow in the manuscript's current form.

    Strengths:

    1. The experiments are generally well designed overall, and appropriate to the questions posed.
    1. Several experimental methods are combined to validate key results.
    1. Use of cutting-edge technologies (i.e. STORM imaging) to help answer key questions in the paper.

    We thank Reviewer #3 for reviewing our manuscript. We sincerely appreciate the valuable feedback. The previous version of the manuscript contained numerous claims, some of which were not thoroughly validated, making it prone to reader misinterpretation. Based on the results of additional experiments, we have revised the manuscript by focusing solely on the findings that were adequately confirmed, specifically highlighting the role of TEN2 in providing a platform for GABAAR exocytosis. We are grateful for your time and effort in revisiting the revised manuscript, and we believe it meets the necessary requirements.

    Weakness:

    1. Simplifying the text and story line would go a long way to ensure the study results are more effectively communicated. Additional specific suggestions are provided in the recommendations for the authors.

    Thank you for providing valuable suggestions. Based on the results of additional experiments, we have revised our claims accordingly.

    1. The introduction overall would benefit from simplification so that the reader is given only the information they need to know to understand the question at hand.

    We selected essential information from previous studies that we believe readers should be aware of before reading our manuscript.

    1. MT dynamics are important for paper results, but the background in the paper does not appropriately introduce this topic.

    We have provided some information in lines 57-64 of the INTRODUCTION section.

    1. It is a bit unclear from the abstract and introduction how the findings of this paper have significantly advanced the field or taught something fundamentally new about how inhibitory synapses are regulated.

    Thank you for your valuable feedback. In the new version, we have thoroughly examined and emphasized the significance of our research findings.

    1. Figure 1 - Line 109, it is obscure why "it was found appropriate" to divide the data into three clusters. This section would better justified by starting with cellular functions and then basing the clusters on these functions.

    As Reviewer #3 pointed out, we have revised the classification to be based on past knowledge rather than data-driven.

    1. The proteomic screen and candidate selection is not well justified and the logic steps for arriving at TEN2 are a bit weak. Again, less is more here.

    As Reviewer #3 mentioned, we have made revisions in the new version. We have not completely excluded NLGN2, but rather believe that further examination and consideration of NLGN2 are necessary going forward (lines 463-471).

    1. Fig. 2 - The authors should consider whether EB1 overexpression would have functional consequences that alter the results and colocalization.

    The previous Figure 2, which is now Figure 6, is intended to demonstrate protein-protein interactions rather than provide functional implications. It is likely that the original function of EB1, which should be located at the plus ends of MTs, is compromised by its presence in the MT lattice. As an alternative method to demonstrate protein-protein interactions, we have also conducted GST pull-down assays (Figure 6A). From these two experimental results, we infer that the intracellular domain of TEN2 interacts with EB1. However, we have not discussed the functional implications of the TEN2-EB1 complex based on these experimental findings. The function was discussed from the results performed in Figure 7.

    1. Fig. 3 - Is immobilization of COS cells using HA tag antibodies a relevant system for study of these questions?

    We agree with this suggestion regarding the replication of the experimental systems to neurons, as the results have been successful in COS-7 cells. However, when we attempted to culture neurons on antibody-coated cover glass, the survival rate was significantly reduced. We were unable to directly replicate these systems to neurons. Therefore, we have decided to withdraw this claim from the publication.

    1. Fig. 4 - The authors should confirm post-synaptic localization in vivo (brain).

    We agree with this suggestion. Currently, our research group does not have an effective immune-labeling method for synaptic protein in the brain. This is a future challenge that we should address.

    1. Figure 4D-E - The way the STORM results are presented is confusing. The authors state is shows that TEN2 is postsynaptic but before this say that the Abs are the same size as the synaptic cleft so that the results cannot be considered conclusive. This issue should be resolved.

    To improve the quality of our dSTORM experiments, we abandon three color dSTORM and instead focused on two color dSTORM to draw conclusions (Figure 3E). We utilized VGAT to detect presynaptic sites. VGAT is an inhibitory presynaptic-specific molecule that is present at the center of presynaptic terminals, eliminating concerns about the size of the antibodies used.

    1. Figure 5 -The authors should examine the levels of gephyrin relative to the levels of knockdown given the knockdown variability.

    Thank you for your suggestion. As shown in Figure 4D of the current version, we were able to simultaneously quantify the knockdown efficiency and synaptic density. We obtained results indicating a decrease in synaptic density associated with a decrease in TEN2 expression levels.

    1. Functional validation of a reduction in inhibition following TEN2 manipulation would elevate the paper.

    We conducted live imaging of EBs to measure the changes when introducing the partial domain of TEN2 (Figures 7A-E). By observing the decrease in synaptic density and the impaired MT recruitment function of endogenous TEN2 due to the dominant-negative effect of TEN2N-L, we concluded that the TEN2-MT interaction serves as the platform for GABAR exocytosis.

    1. Figure 6E - The expression levels of TEN2TM and TEN2NL are important to the outcome of these experiments. How did the authors ensure that the levels of two proteins were the same to begin with?

    As it was also mentioned by Reviewer #1, we reply with the same answer as follows: Regarding the previous co-localization of TEN2 and microtubules after permeabilization with saponin, we have removed it from the analysis because it is not possible to perform accurate quantitative analysis in this case. We speculate that this is a combination of two factors: the variation in transfection efficiency and the inherent variability in permeabilization between neurons. Specifically, it is particularly challenging to standardize and quantify the variability in permeabilization. Instead, the current version proposes TEN2-MT interaction via EBs by live imaging of EB3 in neurons expressing each partial domain. As observed in COS-7 cells where EB was overexpressed, whether TEN2 engages in continuous binding with microtubules or if it is a transient interaction remains an interesting topic for future investigation. We have mentioned this in the DISCUSSION section as well (line 415-422).

  2. Version published to 10.7554/elife.83276 on eLife
  3. eLife

    eLife assessment

    This potentially important paper investigates the mechanisms that contribute to building inhibitory synapses through differential protein release from microtubules. The experiments are generally designed well, but the evidence supporting the conclusions is incomplete. This manuscript will be of interest to neuroscientists and cell biologists interested in intracellular trafficking and synapse maturation.

  4. eLife

    Reviewer #1 (Public Review):

    Ichinose et al., utilize a mixture of cultured hippocampal neurons and non-neuronal cells to identify the role of the transmembrane protein teneurin-2 (TEN-2) in the formation of inhibitory synapses along the dendritic shaft. First, they identify distinct clusters of gephyrin that are either actin-rich, microtubule-rich or contain neither actin nor microtubules and find that TEN-2 is enriched in microtubule-rich gephyrin clusters. This leads the authors to hypothesize that TEN-2 recruits microtubules (MTs) through the plus end binding protein EB1 when successfully matched with a pre-synaptic partner, and perform a variety of experiments to test this hypothesis. The authors then extend this finding to state quite strongly throughout the paper, including in the title, that TEN-2 acts as a signpost for the unloading of cargo from motor proteins without providing any supporting evidence. They use previous work to justify this conclusion, but without actual experiments to back up the claim, it seems like a reach.

    The strength of the paper lies in the various lines of evidence that the authors employ to assess the role of TEN-2 in MT recruitment and synaptogenesis. They have also been very thorough in validating the expression and functionality of various knock-in constructs, knock-down vectors and antibodies that were generated during the study. However, there are some discrepancies in the findings that have not been addressed satisfactorily, as well as some instances where the data presented is not of sufficient quality to support the conclusions derived from them.
    1. The emphasis placed on the clustering analysis presented in figure 1 and the two associated supplementary figures is puzzling, since the conclusion derived from the results presented would be that Neuroligin 2 (NLGN2) is the strongest candidate to test for a relationship to MT recruitment at inhibitory post synapses. Instead, the authors cite prior evidence to exclude NLGN2 from subsequent analysis and choose to focus on TEN2 instead.
    2. It is difficult to reach the same conclusion as the authors from the images and intensity plot shown on Figure 2 E and F. While there seems to be an obvious reduction in expression levels between the TEN2N-L and TEN2TM constructs, neither seem to co-localize with EB1.
    3. The authors mimic the activity of TEN-2 at the inhibitory post synapse in non-neuronal cells by immobilizing HA- tagged TEN constructs in COS-7 cells as a proxy for synaptic partner matching. Using this model, they find that by immobilizing TEN2N-L, which contains EB1 binding motifs, MTs are excluded from the cell periphery (Figure 3D). This contradicts their conclusion that MTs are recruited through EB1 by TEN-2 on synaptic partner matching. Later in the paper, when they use the same TEN2N-L construct as a dominant negative in neuronal cells, they find that MTs are recruited the membrane, even if TEN2N-L is not immobilized by synaptic partner matching (Figure 6C). Taken together, these findings call into question the sequence of events driven by TEN-2 during synaptogenesis.
    4. It is unclear how the authors could conclude that TEN-2 is at the semi-periphery (?) of inhibitory post synapses from the STORM data that is presented in the paper. Figure 4D and 4F show comparisons of Bassoon and TEN-2 localization vs TEN-2 and gephyrin, but the image quality is not sufficient to adequately portray a strong distinction in the distance of center of mass, which is also only depicted for the TEN2-Gephyrin pair and not the TEN2-Bassoon pair in Figure 4J.
    5. The authors do not satisfactorily explain why gephyrin appears to have completely disappeared in the TEN2N-L condition (Figure 6A), instead of appearing uniformly distributed as one would expect if MTs are indiscriminately recruited to the membrane by the dominant negative construct that remains unanchored.
    6. In a similar critique to that of Figure 2E and F, the distinction that the authors wish to portray between the effect of TEN2TM and TEN2N-L constructs on EGFP-TEN-2 and MAP2 colocalization (Figure 6 E and F) appear to be driven by a difference in overall expression levels of EGFP-TEN2 rather that a true difference in localization of TEN-2 and MTs.

  5. eLife

    Reviewer #2 (Public Review):

    Maturation of inhibitory synapses requires multiple vital biological steps including, i) translocation of cargos containing GABAARs and scaffolds (e.g. gephyrin) through microtubules (MTs), ii) exocytosis of inhibitory synapse proteins from cargo followed by the incorporation to the plasma membrane for lateral diffusion, and iii) incorporation of proteins to inhibitory synaptic sites where gephyrin and GABAARs are associated with actin. A number of studies have elucidated the molecular mechanisms for GABAARs and gephyrin translocation in each step. However, the molecular mechanisms underlying the transition between steps, particularly from exocytosis to lateral diffusion of inhibitory proteins, still need to be elucidated. This manuscript successfully characterizes three stages of inhibitory synapses during maturation, cluster1: an initial stage that receptors are being brought in and out by the MT system; cluster2: lateral diffusion stage; cluster 3: matured postsynapses anchored by gephyrin and actin, by quantifying the abundance of MAP2 or Actin in inhibitory synapse labeled by gephyrin. Importantly, the authors' findings suggest that TEN2, a trans-synaptic adhesion molecule that has two EB1 binding motifs, plays an important role in the transition from clusters 1 to 2, and inhibitory synapse maturation. The imaging results are impressive and compelling, these data will provide new insights into the mechanisms of protein transport during synapse development. However, the present study contains several loose ends preventing convincing conclusions. Most importantly, (1) it remains more TEN2 domain characterization on inhibitory synapse maturation, (2) further validation of the HA knock-in TEN2 mouse model is required, and (3) it requires additional physiology data that complement the authors' findings.

  6. eLife

    Reviewer #3 (Public Review):

    In this paper, Ichinose et al. examine mechanisms that contribute to building inhibitory synapses through differential protein release from microtubules. They find that tenurin-2 plays a role in this process in cultured hippocampal neurons via EB1 using a variety of genetic and imaging methods. Overall, the experiments are generally designed well, but it is unclear whether their findings offer a significant advance. The experimental logic flow and rational difficult for readers to follow in the manuscript's current form.

    Strengths:

    1. The experiments are generally well designed overall, and appropriate to the questions posed.
    2. Several experimental methods are combined to validate key results.
    3. Use of cutting-edge technologies (i.e. STORM imaging) to help answer key questions in the paper.

    Weakness:

    1. Simplifying the text and story line would go a long way to ensure the study results are more effectively communicated. Additional specific suggestions are provided in the recommendations for the authors.
    2. The introduction overall would benefit from simplification so that the reader is given only the information they need to know to understand the question at hand.
    3. MT dynamics are important for paper results, but the background in the paper does not appropriately introduce this topic.
    4. It is a bit unclear from the abstract and introduction how the findings of this paper have significantly advanced the field or taught something fundamentally new about how inhibitory synapses are regulated.
    5. Figure 1 - Line 109, it is obscure why "it was found appropriate" to divide the data into three clusters. This section would better justified by starting with cellular functions and then basing the clusters on these functions.
    6. The proteomic screen and candidate selection is not well justified and the logic steps for arriving at TEN2 are a bit weak. Again, less is more here.
    7. Fig. 2 - The authors should consider whether EB1 overexpression would have functional consequences that alter the results and colocalization.
    8. Fig. 3 - Is immobilization of COS cells using HA tag antibodies a relevant system for study of these questions?
    9. Fig. 4 - The authors should confirm post-synaptic localization in vivo (brain).
    10. Figure 4D-E - The way the STORM results are presented is confusing. The authors state is shows that TEN2 is postsynaptic but before this say that the Abs are the same size as the synaptic cleft so that the results cannot be considered conclusive. This issue should be resolved.
    11. Figure 5 -The authors should examine the levels of gephyrin relative to the levels of knockdown given the knockdown variability.
    12. Functional validation of a reduction in inhibition following TEN2 manipulation would elevate the paper.
    13. Figure 6E - The expression levels of TEN2TM and TEN2NL are important to the outcome of these experiments. How did the authors ensure that the levels of two proteins were the same to begin with?
  7. Version published to 10.1101/2022.09.13.507723v1 on bioRxiv