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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Synapses are the sites that mediate chemical signal transduction from axons to the dendrites of other neurons. At the axonal side of the synapse, called the presynapse, the arrival of action potentials triggers the fusion of synaptic vesicles filled with neurotransmitters with the plasma membrane. These neurotransmitters will then be detected by receptors located at the post-synapse at the dendrite.

    In this manuscript, the authors set out to study the nanoarchitecture of the actin cytoskeleton at presynapses. They argue that this is challenging due to the much higher density of actin at the postsynapse. Therefore, they use an established approach in which polylysine-coated beads can induce structures at the axon that resemble presynapses. The authors first further characterize these presynapse-like structures by measuring the intensity of different presynaptic proteins. They report the presence bassoon, synapsin, synoptophysin, vamp2 at levels that are about half the level found at real synapses (Figure 1). 67% of induced hemisynapses are enriched for actin and the level of synaptic markers is higher in the presence of actin (Figure 2). Furthermore, actin-enriched hemisynapses also display more vesicle recycling than induced hemisynapses without actin enrichment.

    Next, the intensity of synaptic markers is measured after treatment with drugs that stabilize or destabilize actin (Fig. 4), or drugs that inhibit the actin nucleators Arp2/3 or Formin (Fig. 5), which reveals some differences that could suggest the existence of different types of actin-based assemblies. The super-resolution microscopy in Figures 6 and 7 indeed nicely demonstrate that existence of actin as either dense clouds or clearly resolved fibers. Overall, this is an interesting manuscript that uses a simplified model system to study the organization of actin at presynapse-like structures. The super-resolution images provide exciting evidence for the existence of distinct actin structures in different parts of the presynapse, which provides many avenues for further research. Overall, the data appear solid and well-quantified. However, I do have a number of comments about the relevance of the model system and the presentation and interpretation of the data.

    Major comments

    Model system

    1. The authors use the bead-triggered formation of synaptic-like presynaptic structures, because they argue that the post-synaptic actin would overwhelm any actin signal from the presynapses. This is demonstrated in Figure S1, where the authors use 20 div neurons and show that post-synaptic actin is brighter than presynaptic actin. However, this demonstration raises a number of questions. Why did they authors demonstrate this with 20 div neurons, whereas the rest of the manuscript focusses on neurons that are much younger (9 div)? These younger neurons typically have much less dendritic spines and the cultures are easier to navigate due to the lower density of axons. In their examples, the authors also mostly highlight excitatory synapses located at actin-rich dendritic spines and it is not directly evident that this is also true for inhibitory synapses that typically connect to the dendritic shaft. For example, the example shown in Figure 6B suggests that actin density is higher at the pre-synapse than at the post-synapse. According to the authors 20-40% of synapses in their culture are inhibitory synapses, so I would encourage the authors to try to get more data on real synapses, perhaps at 9 div. In my view, demonstration of the existence of the proposed actin structure at bonafide synapses would make the author's claims much stronger.
    2. Related to the earlier point, the authors also acknowledge that alternative approaches, such as expression of lifeAct or GFP-actin would be possible to probe presynaptic actin organization at real synapses, but that these constructs can only be used at low levels in order to prevent artefacts. While in principle this is correct, recent successes in establishing knock-in approaches in differentiated neurons (i.e. HITI, ORANGE) have shown that endogenous actin can be tagged with small tags. Therefore knock-in of small epitope tags, such as HA or ALFA, would be a relatively straightforward way to selectively label presynaptic actin in real synapses. As mentioned above, demonstration of the existence of the proposed actin structure at bonafide synapses would make the author's claims much stronger.
    3. The authors show that various key presynaptic proteins are about half as abundant on the synaptic-like presynaptic structure compared to real synapses. They argue that this might reflect the fact that the bead-induced synapse-like structures were analyzed two days after addition of the beads, whereas the real synapses might already have matured longer. This could easily be tested by altering the incubation time of beads and/or by analyzing how the average intensity of synapses develops over time. In addition, it is important to know how the intensity of actin compares between real synapses (NS) and induced synapses, because some images suggests that the enrichment at induced synapses is higher than at real synapses. This could suggest that the actin structures found at induced synapses might be specific to these induced hemisynapses. Data presentation
    4. In Figure 1, the authors classify induced hemisynapses as either enriched for actin or not and then move on to analyze the intensity of bassoon, synapsin, synoptophysin, vamp2 for the two classes of hemisynapses. This promotes a very binary view of the structures they induce, whereas I assume that the intensity of actin will vary from structure to structure. Therefore, it would be more useful to plot the intensity of bassoon, synapsin, synoptophysin, vamp2 as a function of the intensity of actin. This could reveal that there are two clear regimes, but a least that would provide a justification for the classification into A+ and A-.
    5. In Figure 3, vesicular cycling is compared between actin-enriched and non-enriched induced hemisynapses. It would be good to include a comparison with real synapses.

    Biological interpretation

    1. The title of the manuscript is "Distinct nano-structures support a multifunctional role of actin at presynapses". I agree that the identification of distinct structures supports the idea that they have distinct functions, but I do not think that the current manuscript really demonstrates that the distinct nano-structures support different roles. The result that actin stabilization and disassembly both affect vesicular cycling is taken as support for the idea that distinct actin structures coexist within the presynapse. In my view, it mostly demonstrates that a dynamic actin cytoskeleton is needed for vesicular cycling. Given the role of actin dynamics in endocytosis, this is not really a surprise. Likewise, the authors interpret the experiments in Fig. 5, where different actin nucleators are inhibited, as further evidence for distinct presynaptic structures. Although these might well exist, I am not sure if these experiment reveal that. Inhibition of Arp2/3 has very little effect, whereas inhibition of formins leads to more actin. Overall, these pharmacological experiments are very hard to interpret and do not directly promote the idea that different nucleators generate presynaptic actin networks with distinct functions.
    2. The imaging in Figure 6 and 7 is very nice and does provide new insights into the organization of actin at induced hemi-synapses. While I certainly do understand the desire to name these structures, it is currently not clear what the structural difference would be between an actin mesh and an actin corral, and between an actin rail and an actin trail. Intuitively, one would think that meshes and corrals are generated by Arp2/3 based nucleation, while rails and trails are generate by formins. However, the analysis in Figure 7 does not really support this thinking. It could be that the quantification in Figure 7 a bit too coarse grained, because it mostly looks if structures are present or not. A more subtle analysis would analyze the intensities or sizes of meshes, rails and corrals and plot those in different conditions. Did the authors try something like that?
    3. I do agree with the speculation that corrals could be used to confine vesicles (and perhaps to fish them out of the flow of axonal transport by actin-binding tethering factors), while the rails could facilitate local transport to the active zone. While the authors hypothesize that the actin mesh could inhibit vesicle release, another option is that it promotes endocytosis.

    Questions related to the major comment

    • Are the key conclusions convincing?

    The data is convincing, some of the data is over-interpreted.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    See my specific comments above.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    It would be fantastic if the authors can provide evidence for the structures they describe by the analysis of real synapses, for example by using knock-in approaches. Without additional data, the authors should reconsider some of their claims and interpretations and provide a more balanced discussion.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    It is my understanding that the team has successfully achieved endogenous tagging of actin, but they might have good reasons for not using it for the current story.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Most procedures are well-documented. Some of the classification strategies are not extensively outlined.

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes, the supplemental tables with replicate number etc is very useful.

    Minor comments

    1. In Figure 4, the effect of actin destabilization or stabilization on the level of synaptic proteins is examined. Here the axis labels are a bit confusing. The label "swin A-" suggests that there were also swin A+ synapses that were not analyzed. Similarly, the cuc A+ label suggest the exclusion of cuc A- synapses. Were there still A+ / A- synapses upon treatment of swin/cuc, respectively? If so, what happened to the relative abundance of A+/A- in these conditions?
    2. The last paragraph of the result section should be part of the discussion section.

    Referees cross-commenting

    Overall, all three reviewer provide very similar feedback.

    • a need for more careful interpretation of the induced structures and their relevance to real synapses.
    • more characterization of the induced hemi-synapses in terms of localization (mostly on axons), actin density compared to real synapses, intensity of synaptic proteins at different days after induction, etc. A key concern is that the identified actin structures are specific for these induced structures.
    • a need for more careful interpretation of the effects of the various drug treatments, as well as the formation and function of the various actin structures
    • an encouragement to try to selectively label presynaptic actin using genetic approaches

    Significance

    This work provides exciting evidence for the existence of distinct actin structures in different parts of the presynapse, which provides many avenues for further research. While the structure and dynamics of the presynapse has been studied for decades, little is known about the organization of the actin cytoskeleton at these key sites of neuronal signal transmission.

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    Referee #2

    Evidence, reproducibility and clarity

    Bingham et al., studied the composition and nano-structure of presynaptic elements induced by polylysine beads. By isolating the presynaptic element from the actin-rich postsynaptic compartment, this system makes it possible to study the organization of actin structures at high resolution. Using a combination of pharmacological interventions and super-resolution imaging, the authors distinguish three different types of actin structures at presynaptic elements.

    Overall, this is a very careful study, using an innovative approach and state-of-the-art imaging techniques to visualize actin structures in presynaptic structures. The characterization of bead-induced presynaptic structures is elaborate and provides support that these structures can be used as a proxy of 'natural' synapses. The imaging, particularly the super-resolution imaging if of very high standard and convincing. The writing is overall clear and pleasant to read. Nevertheless, a number of concerns prevent strong conclusions from this study about how different actin structures support the structure and function of natural synapses.

    • The characterization of bead-induced structures is quite extensive and also literature suggests that these structures are 'functional' in the sense that vesicle recycling çan be detected. Nevertheless, from the images it is not clear that these structures are always formed on axons. It seems the beads also induce presynaptic elements on dendrites, which would be highly artificial and prevent strong conclusions about axonal actin organization. Can the authors provide support that these "presynaptic structures" are preferentially formed on axons?
    • The key observation of this paper is the existence of distinct actin structures highlighted in figures 6 and 7. These structures are indeed distinguishable by eye and could be of interest, but it remains unclear how these were defined. This is not described in the methods section, which makes it difficult to interpret the value of this observation. Were any (quantitative) criteria defined to outline these structures?
    • The pharmacological intervention experiments in Figure 7D show modest, non-significant effects. More support that these structures are truly distinct and functional is required or conclusions about the existence of distinct actin assemblies should be reworded. Also see points below.
    • A main concern is to what extent the contacts with the bead induce specific actin structures that are not representative of actin structures in natural synapses. The artificial, strong recruitment of heparan sulfate glycans could potentially induce the clustering of all kinds of adhesion complexes that promote actin polymerization/branching etc. and overrules the fine scale distribution of adhesion molecules and other presynaptic proteins in natural synapses. It thus remains unclear how specific and relevant these actin assemblies are for synapses. When comparing the natural synapse and induced synapse in Figure 6B and C it seems that particularly the 'actin rails' seem to originate from the bead contact (while similar structures cannot be seen in the natural synapse) and could thus reflect strong actin polymerization induced simply by the contact with the bead. More support that the observation of distinct actin structures is reminiscent of structures found at natural synapses is required. Experiments to show that such structures for instance do not form on non-neuronal cells could be considered. Experiments at natural synapses would of course be preferred. Have the authors considered genetic approaches to label actin in isolated cells? In that manner the presynaptic compartment could also easily be distinguished from the postsynaptic dendritic spines. A number of actin reporters (LifeAct, Ftractin, Utrophin, etc) are available, and albeit these have their limitations, if carefully used, these could be used to demonstrate similar structures. Alternatively, several CRISPR/Cas9 genome editing approaches are now available (HiUGE, ORANGE, TKIT, CRISPIE) that enable visualization of endogenous actin in isolated neurons.
    • Since actin structures are responding to changes in neuronal activity, the (selective) modulation of these three types of actin assemblies to short- and/or long-term changes in neuronal activity would be of great interest and help support the functional relevance of this observation.

    Minor:

    • The term "presynapse" is not very commonly used in literature to indicate the presynaptic compartment. Particularly in this case it is a bit misleading as it suggests there is also a corresponding postsynaptic element. I would recommend to use 'presynaptic compartment' or alike.
    • Reference to Glebov et al., Cell Reports 2017 is missing, even though this is a highly relevant study using SMLM to study active zone organization and the role of actin dynamics in regulating AZ composition.
    • The labels in the images with the purple font on the black background (e.g., "Bassoon" in Figure 1A) are hardly visible
    • The graphs should include an indication of statistical significance
    • The term "concentration" is sometimes used when intensity measurements are done, but that is not appropriate in that case and should be rephrased to e.g. "relative amount" or alike.
    • In abstract: "dependance" > "dependence
    • Page 1: "Decade of research" > "Decades of research
    • Page 4: "not at high" > "not as high"
    • On page 10, "dependant" > "dependent'
    • On page 14, "recruitment of neuroligin1" , the authors mean "neurexin1"?

    Referees cross-commenting

    I agree with the comments of the other reviewers, I see overall very similar comments. This is a strong and valuable study, but the main conclusions need more experimental support. Particularly, more quantitative characterisation of the induced synapses is needed and more support that the proposed classification of actin structures is representative of structures found in physiological synapses. For the last point, genetic labelling of actin structures in physiologic synapses is indeed strongly encouraged as also indicated by reviewer #3.

    Significance

    This study provides a detailed characterization of bead-induced presynaptic structures that allow the investigation of presynaptic actin structures at unprecedented resolution. The authors suggest that the presence of distinct actin structures at presynaptic specializations serve different functions to sustain synaptic transmission. These findings are of great interest for molecular and cellular neuroscientists interested in presynaptic mechanisms, but also more generally audience interested in super-resolution microscopy and/or the actin cytoskeleton.

    I have experience in molecular and cellular neuroscience, synaptic transmission, and diverse microscopy techniques.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    While phalloidin has been widely used to stain actin, its major limitation is that it labels both the presynaptic and postsynaptic actin structures, making it difficult to properly the comparatively less dense actin characterize within the presynapse. To overcome these difficulties, Bingham et al. made use of the well-established presynaptic induction model that utilises polylysine-coated beads to induce rapid formation of functionals synapses. They apply wide field fluorescence imaging showing that actin is enriched in these bead-induced synapses and apply actin nucleation and polymerisation inhibitors to characterise essential role of actin in maintaining the levels of other presynaptic components. Further, they apply nanoscale STORM and PAINT imaging to uncover distinct actin structures within the presynapse and how this is regulated by nucleation mechanisms.

    Major Comments

    • After the seeding beads in DIV 9-10 neurons the authors fix the neurons 48 hours later and indicate that they have functional synapses (S+) with protein presynaptic protein intensity less than natural synapses (Fig. 1). A key argument made by the authors is that the natural synapses are older than the bead-induced S+ ones. The expectation therefore then is that if the fix the neurons 72 or 96 hours after bead treatment, then the S+ should have a higher intensity than synapses after 48 hours. The authors should provide a time graded increase in synaptic component intensity to solidify their argument.
    • Based on Fig.2 and Fig.3, the authors indicate actin enrichment in a subset of bead-induced synapses. The authors however did not provide a reasoning for why there is no actin enrichment in up to 30% of beads-induced synapses.
    • Does shorter time treatment (for example 30 mins) of the induced synapses with swinholide and cucurbitacin E similarly reduce the intensity of presynaptic components?
    • Using the CK666 actin nucleation inhibitor, the authors should provide supplemental information of no changes in intensity to other synaptic vesicle proteins (for example SV2) and to that of other presynaptic plasma membrane proteins such as Syntaxin-1 and Munc13.
    • The authors should expand their STORM experiments to verify other data acquired with wide field fluorescence microscope such as actin enrichment (Fig.2) in bead-induced synapses

    Minor Comments

    • The authors should cite Rust et al., 2006 Nat. Methods as reference to first mention of STORM in paragraph 2 of the introduction.
    • In Fig.S1, the authors indicate the dashed yellow lines as the presynapse. A better label, that stains the entire length of the presynapse might be needed to convincingly indicate presynaptic actin (dashed yellow lines) outside the bassoon labelling.
    • The authors should provide quantification for the FM1-43 dye loading experiments in Fig.S2E and F.
    • The author should provide representative images for the data from natural synapses in Fig.S5 for control, swinholide A, cucurbitacin E, CK666 and SMIFH2 treatments.

    Referees cross-commenting

    I agree with the comments from the other two reviewers that more work needs to be done to sufficiently justify the conclusions made.

    Significance

    • A key highlight that Bingham et al brings to the field is that they push the field forward from previous classical work done by the Zhuang lab (Xu et al., 2013 Science) where they showed novel data on the periodic organisation of actin cytoskeleton. This was done especially by provided a mechanism (bead induced synapse production) to narrow down on viewing presynaptic actin without overlapping 'noise' from postsynaptic region.
    • Applying multiple nanoscale advanced imaging (STORM and PAINT) also helped solidify their data and provide hitherto unseen characterisation of actin structures.
    • This manuscript will provide key insight to all scientists in the field of cell biology and cancer research that work on precisely characterising the cytoskeletal structure of the cell.
    • Key words of field of expertise: Super-resolution microscopy, Neuroscience, Dementia, Synapse, Drosophila
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    Reply to the reviewers

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Synapses are the sites that mediate chemical signal transduction from axons to the dendrites of other neurons. At the axonal side of the synapse, called the presynapse, the arrival of action potentials triggers the fusion of synaptic vesicles filled with neurotransmitters with the plasma membrane. These neurotransmitters will then be detected by receptors located at the post-synapse at the dendrite.

    In this manuscript, the authors set out to study the nanoarchitecture of the actin cytoskeleton at presynapses. They argue that this is challenging due to the much higher density of actin at the postsynapse. Therefore, they use an established approach in which polylysine-coated beads can induce structures at the axon that resemble presynapses. The authors first further characterize these presynapse-like structures by measuring the intensity of different presynaptic proteins. They report the presence bassoon, synapsin, synoptophysin, vamp2 at levels that are about half the level found at real synapses (Figure 1). 67% of induced hemisynapses are enriched for actin and the level of synaptic markers is higher in the presence of actin (Figure 2). Furthermore, actin-enriched hemisynapses also display more vesicle recycling than induced hemisynapses without actin enrichment.

    Next, the intensity of synaptic markers is measured after treatment with drugs that stabilize or destabilize actin (Fig. 4), or drugs that inhibit the actin nucleators Arp2/3 or Formin (Fig. 5), which reveals some differences that could suggest the existence of different types of actin-based assemblies. The super-resolution microscopy in Figures 6 and 7 indeed nicely demonstrate that existence of actin as either dense clouds or clearly resolved fibers. Overall, this is an interesting manuscript that uses a simplified model system to study the organization of actin at presynapse-like structures. The super-resolution images provide exciting evidence for the existence of distinct actin structures in different parts of the presynapse, which provides many avenues for further research. Overall, the data appear solid and well-quantified. However, I do have a number of comments about the relevance of the model system and the presentation and interpretation of the data.

    Major comments

    Model system

    1. The authors use the bead-triggered formation of synaptic-like presynaptic structures, because they argue that the post-synaptic actin would overwhelm any actin signal from the presynapses. This is demonstrated in Figure S1, where the authors use 20 div neurons and show that post-synaptic actin is brighter than presynaptic actin. However, this demonstration raises a number of questions. Why did they authors demonstrate this with 20 div neurons, whereas the rest of the manuscript focusses on neurons that are much younger (9 div)? These younger neurons typically have much less dendritic spines and the cultures are easier to navigate due to the lower density of axons. In their examples, the authors also mostly highlight excitatory synapses located at actin-rich dendritic spines and it is not directly evident that this is also true for inhibitory synapses that typically connect to the dendritic shaft. For example, the example shown in Figure 6B suggests that actin density is higher at the pre-synapse than at the post-synapse. According to the authors 20-40% of synapses in their culture are inhibitory synapses, so I would encourage the authors to try to get more data on real synapses, perhaps at 9 div. In my view, demonstration of the existence of the proposed actin structure at bonafide synapses would make the author's claims much stronger.
    2. Related to the earlier point, the authors also acknowledge that alternative approaches, such as expression of lifeAct or GFP-actin would be possible to probe presynaptic actin organization at real synapses, but that these constructs can only be used at low levels in order to prevent artefacts. While in principle this is correct, recent successes in establishing knock-in approaches in differentiated neurons (i.e. HITI, ORANGE) have shown that endogenous actin can be tagged with small tags. Therefore knock-in of small epitope tags, such as HA or ALFA, would be a relatively straightforward way to selectively label presynaptic actin in real synapses. As mentioned above, demonstration of the existence of the proposed actin structure at bonafide synapses would make the author's claims much stronger.
    3. The authors show that various key presynaptic proteins are about half as abundant on the synaptic-like presynaptic structure compared to real synapses. They argue that this might reflect the fact that the bead-induced synapse-like structures were analyzed two days after addition of the beads, whereas the real synapses might already have matured longer. This could easily be tested by altering the incubation time of beads and/or by analyzing how the average intensity of synapses develops over time. In addition, it is important to know how the intensity of actin compares between real synapses (NS) and induced synapses, because some images suggests that the enrichment at induced synapses is higher than at real synapses. This could suggest that the actin structures found at induced synapses might be specific to these induced hemisynapses. Data presentation
    4. In Figure 1, the authors classify induced hemisynapses as either enriched for actin or not and then move on to analyze the intensity of bassoon, synapsin, synoptophysin, vamp2 for the two classes of hemisynapses. This promotes a very binary view of the structures they induce, whereas I assume that the intensity of actin will vary from structure to structure. Therefore, it would be more useful to plot the intensity of bassoon, synapsin, synoptophysin, vamp2 as a function of the intensity of actin. This could reveal that there are two clear regimes, but a least that would provide a justification for the classification into A+ and A-.
    5. In Figure 3, vesicular cycling is compared between actin-enriched and non-enriched induced hemisynapses. It would be good to include a comparison with real synapses.

    Biological interpretation

    1. The title of the manuscript is "Distinct nano-structures support a multifunctional role of actin at presynapses". I agree that the identification of distinct structures supports the idea that they have distinct functions, but I do not think that the current manuscript really demonstrates that the distinct nano-structures support different roles. The result that actin stabilization and disassembly both affect vesicular cycling is taken as support for the idea that distinct actin structures coexist within the presynapse. In my view, it mostly demonstrates that a dynamic actin cytoskeleton is needed for vesicular cycling. Given the role of actin dynamics in endocytosis, this is not really a surprise. Likewise, the authors interpret the experiments in Fig. 5, where different actin nucleators are inhibited, as further evidence for distinct presynaptic structures. Although these might well exist, I am not sure if these experiment reveal that. Inhibition of Arp2/3 has very little effect, whereas inhibition of formins leads to more actin. Overall, these pharmacological experiments are very hard to interpret and do not directly promote the idea that different nucleators generate presynaptic actin networks with distinct functions.
    2. The imaging in Figure 6 and 7 is very nice and does provide new insights into the organization of actin at induced hemi-synapses. While I certainly do understand the desire to name these structures, it is currently not clear what the structural difference would be between an actin mesh and an actin corral, and between an actin rail and an actin trail. Intuitively, one would think that meshes and corrals are generated by Arp2/3 based nucleation, while rails and trails are generate by formins. However, the analysis in Figure 7 does not really support this thinking. It could be that the quantification in Figure 7 a bit too coarse grained, because it mostly looks if structures are present or not. A more subtle analysis would analyze the intensities or sizes of meshes, rails and corrals and plot those in different conditions. Did the authors try something like that?
    3. I do agree with the speculation that corrals could be used to confine vesicles (and perhaps to fish them out of the flow of axonal transport by actin-binding tethering factors), while the rails could facilitate local transport to the active zone. While the authors hypothesize that the actin mesh could inhibit vesicle release, another option is that it promotes endocytosis.

    Questions related to the major comment

    • Are the key conclusions convincing?

    The data is convincing, some of the data is over-interpreted.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    See my specific comments above.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    It would be fantastic if the authors can provide evidence for the structures they describe by the analysis of real synapses, for example by using knock-in approaches. Without additional data, the authors should reconsider some of their claims and interpretations and provide a more balanced discussion.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    It is my understanding that the team has successfully achieved endogenous tagging of actin, but they might have good reasons for not using it for the current story.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Most procedures are well-documented. Some of the classification strategies are not extensively outlined.

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes, the supplemental tables with replicate number etc is very useful.

    Minor comments

    1. In Figure 4, the effect of actin destabilization or stabilization on the level of synaptic proteins is examined. Here the axis labels are a bit confusing. The label "swin A-" suggests that there were also swin A+ synapses that were not analyzed. Similarly, the cuc A+ label suggest the exclusion of cuc A- synapses. Were there still A+ / A- synapses upon treatment of swin/cuc, respectively? If so, what happened to the relative abundance of A+/A- in these conditions?
    2. The last paragraph of the result section should be part of the discussion section.

    Referees cross-commenting

    Overall, all three reviewer provide very similar feedback.

    • a need for more careful interpretation of the induced structures and their relevance to real synapses.
    • more characterization of the induced hemi-synapses in terms of localization (mostly on axons), actin density compared to real synapses, intensity of synaptic proteins at different days after induction, etc. A key concern is that the identified actin structures are specific for these induced structures.
    • a need for more careful interpretation of the effects of the various drug treatments, as well as the formation and function of the various actin structures
    • an encouragement to try to selectively label presynaptic actin using genetic approaches

    Significance

    This work provides exciting evidence for the existence of distinct actin structures in different parts of the presynapse, which provides many avenues for further research. While the structure and dynamics of the presynapse has been studied for decades, little is known about the organization of the actin cytoskeleton at these key sites of neuronal signal transmission.

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    Referee #2

    Evidence, reproducibility and clarity

    Bingham et al., studied the composition and nano-structure of presynaptic elements induced by polylysine beads. By isolating the presynaptic element from the actin-rich postsynaptic compartment, this system makes it possible to study the organization of actin structures at high resolution. Using a combination of pharmacological interventions and super-resolution imaging, the authors distinguish three different types of actin structures at presynaptic elements.

    Overall, this is a very careful study, using an innovative approach and state-of-the-art imaging techniques to visualize actin structures in presynaptic structures. The characterization of bead-induced presynaptic structures is elaborate and provides support that these structures can be used as a proxy of 'natural' synapses. The imaging, particularly the super-resolution imaging if of very high standard and convincing. The writing is overall clear and pleasant to read. Nevertheless, a number of concerns prevent strong conclusions from this study about how different actin structures support the structure and function of natural synapses.

    • The characterization of bead-induced structures is quite extensive and also literature suggests that these structures are 'functional' in the sense that vesicle recycling çan be detected. Nevertheless, from the images it is not clear that these structures are always formed on axons. It seems the beads also induce presynaptic elements on dendrites, which would be highly artificial and prevent strong conclusions about axonal actin organization. Can the authors provide support that these "presynaptic structures" are preferentially formed on axons?
    • The key observation of this paper is the existence of distinct actin structures highlighted in figures 6 and 7. These structures are indeed distinguishable by eye and could be of interest, but it remains unclear how these were defined. This is not described in the methods section, which makes it difficult to interpret the value of this observation. Were any (quantitative) criteria defined to outline these structures?
    • The pharmacological intervention experiments in Figure 7D show modest, non-significant effects. More support that these structures are truly distinct and functional is required or conclusions about the existence of distinct actin assemblies should be reworded. Also see points below.
    • A main concern is to what extent the contacts with the bead induce specific actin structures that are not representative of actin structures in natural synapses. The artificial, strong recruitment of heparan sulfate glycans could potentially induce the clustering of all kinds of adhesion complexes that promote actin polymerization/branching etc. and overrules the fine scale distribution of adhesion molecules and other presynaptic proteins in natural synapses. It thus remains unclear how specific and relevant these actin assemblies are for synapses. When comparing the natural synapse and induced synapse in Figure 6B and C it seems that particularly the 'actin rails' seem to originate from the bead contact (while similar structures cannot be seen in the natural synapse) and could thus reflect strong actin polymerization induced simply by the contact with the bead. More support that the observation of distinct actin structures is reminiscent of structures found at natural synapses is required. Experiments to show that such structures for instance do not form on non-neuronal cells could be considered. Experiments at natural synapses would of course be preferred. Have the authors considered genetic approaches to label actin in isolated cells? In that manner the presynaptic compartment could also easily be distinguished from the postsynaptic dendritic spines. A number of actin reporters (LifeAct, Ftractin, Utrophin, etc) are available, and albeit these have their limitations, if carefully used, these could be used to demonstrate similar structures. Alternatively, several CRISPR/Cas9 genome editing approaches are now available (HiUGE, ORANGE, TKIT, CRISPIE) that enable visualization of endogenous actin in isolated neurons.
    • Since actin structures are responding to changes in neuronal activity, the (selective) modulation of these three types of actin assemblies to short- and/or long-term changes in neuronal activity would be of great interest and help support the functional relevance of this observation.

    Minor:

    • The term "presynapse" is not very commonly used in literature to indicate the presynaptic compartment. Particularly in this case it is a bit misleading as it suggests there is also a corresponding postsynaptic element. I would recommend to use 'presynaptic compartment' or alike.
    • Reference to Glebov et al., Cell Reports 2017 is missing, even though this is a highly relevant study using SMLM to study active zone organization and the role of actin dynamics in regulating AZ composition.
    • The labels in the images with the purple font on the black background (e.g., "Bassoon" in Figure 1A) are hardly visible
    • The graphs should include an indication of statistical significance
    • The term "concentration" is sometimes used when intensity measurements are done, but that is not appropriate in that case and should be rephrased to e.g. "relative amount" or alike.
    • In abstract: "dependance" > "dependence
    • Page 1: "Decade of research" > "Decades of research
    • Page 4: "not at high" > "not as high"
    • On page 10, "dependant" > "dependent'
    • On page 14, "recruitment of neuroligin1" , the authors mean "neurexin1"?

    Referees cross-commenting

    I agree with the comments of the other reviewers, I see overall very similar comments. This is a strong and valuable study, but the main conclusions need more experimental support. Particularly, more quantitative characterisation of the induced synapses is needed and more support that the proposed classification of actin structures is representative of structures found in physiological synapses. For the last point, genetic labelling of actin structures in physiologic synapses is indeed strongly encouraged as also indicated by reviewer #3.

    Significance

    This study provides a detailed characterization of bead-induced presynaptic structures that allow the investigation of presynaptic actin structures at unprecedented resolution. The authors suggest that the presence of distinct actin structures at presynaptic specializations serve different functions to sustain synaptic transmission. These findings are of great interest for molecular and cellular neuroscientists interested in presynaptic mechanisms, but also more generally audience interested in super-resolution microscopy and/or the actin cytoskeleton.

    I have experience in molecular and cellular neuroscience, synaptic transmission, and diverse microscopy techniques.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    While phalloidin has been widely used to stain actin, its major limitation is that it labels both the presynaptic and postsynaptic actin structures, making it difficult to properly the comparatively less dense actin characterize within the presynapse. To overcome these difficulties, Bingham et al. made use of the well-established presynaptic induction model that utilises polylysine-coated beads to induce rapid formation of functionals synapses. They apply wide field fluorescence imaging showing that actin is enriched in these bead-induced synapses and apply actin nucleation and polymerisation inhibitors to characterise essential role of actin in maintaining the levels of other presynaptic components. Further, they apply nanoscale STORM and PAINT imaging to uncover distinct actin structures within the presynapse and how this is regulated by nucleation mechanisms.

    Major Comments

    • After the seeding beads in DIV 9-10 neurons the authors fix the neurons 48 hours later and indicate that they have functional synapses (S+) with protein presynaptic protein intensity less than natural synapses (Fig. 1). A key argument made by the authors is that the natural synapses are older than the bead-induced S+ ones. The expectation therefore then is that if the fix the neurons 72 or 96 hours after bead treatment, then the S+ should have a higher intensity than synapses after 48 hours. The authors should provide a time graded increase in synaptic component intensity to solidify their argument.
    • Based on Fig.2 and Fig.3, the authors indicate actin enrichment in a subset of bead-induced synapses. The authors however did not provide a reasoning for why there is no actin enrichment in up to 30% of beads-induced synapses.
    • Does shorter time treatment (for example 30 mins) of the induced synapses with swinholide and cucurbitacin E similarly reduce the intensity of presynaptic components?
    • Using the CK666 actin nucleation inhibitor, the authors should provide supplemental information of no changes in intensity to other synaptic vesicle proteins (for example SV2) and to that of other presynaptic plasma membrane proteins such as Syntaxin-1 and Munc13.
    • The authors should expand their STORM experiments to verify other data acquired with wide field fluorescence microscope such as actin enrichment (Fig.2) in bead-induced synapses

    Minor Comments

    • The authors should cite Rust et al., 2006 Nat. Methods as reference to first mention of STORM in paragraph 2 of the introduction.
    • In Fig.S1, the authors indicate the dashed yellow lines as the presynapse. A better label, that stains the entire length of the presynapse might be needed to convincingly indicate presynaptic actin (dashed yellow lines) outside the bassoon labelling.
    • The authors should provide quantification for the FM1-43 dye loading experiments in Fig.S2E and F.
    • The author should provide representative images for the data from natural synapses in Fig.S5 for control, swinholide A, cucurbitacin E, CK666 and SMIFH2 treatments.

    Referees cross-commenting

    I agree with the comments from the other two reviewers that more work needs to be done to sufficiently justify the conclusions made.

    Significance

    • A key highlight that Bingham et al brings to the field is that they push the field forward from previous classical work done by the Zhuang lab (Xu et al., 2013 Science) where they showed novel data on the periodic organisation of actin cytoskeleton. This was done especially by provided a mechanism (bead induced synapse production) to narrow down on viewing presynaptic actin without overlapping 'noise' from postsynaptic region.
    • Applying multiple nanoscale advanced imaging (STORM and PAINT) also helped solidify their data and provide hitherto unseen characterisation of actin structures.
    • This manuscript will provide key insight to all scientists in the field of cell biology and cancer research that work on precisely characterising the cytoskeletal structure of the cell.
    • Key words of field of expertise: Super-resolution microscopy, Neuroscience, Dementia, Synapse, Drosophila
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