Non-muscle myosin II regulates presynaptic actin assemblies and neuronal mechanobiology in Drosophila

  1. Biljana Ermanoska
  2. Jonathan Baets
  3. Avital Adah Rodal

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Abstract

Neuromuscular junctions (NMJs) are evolutionarily ancient, specialized contacts between neurons and muscles. They endure mechanical strain from muscle contractions throughout life, but cellular mechanisms for managing this stress remain unclear. Here we identify a novel actomyosin structure at Drosophila larval NMJs, consisting of a long-lived, low-turnover presynaptic actin core that co-localizes with non-muscle myosin II (NMII). This core is likely to have contractile properties, as manipulating neuronal NMII levels or activity disrupts its organization. Intriguingly, depleting neuronal NMII triggered changes in postsynaptic muscle NMII levels and organization near synapses, suggesting transsynaptic propagation of actomyosin rearrangements. We also found reduced levels of Integrin adhesion receptors both pre- and postsynaptically upon NMII knockdown, indicating disrupted neuron-muscle connections. Mechanical severing of axons caused similar actin core fragmentation and Integrin loss to NMII depletion, suggesting this structure responds to tension. Our findings reveal a presynaptic actomyosin assembly that maintains mechanical continuity between neurons and muscle, possibly facilitating mechanotransduction at the NMJ via Integrin-mediated adhesion.

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  1. Version published to 10.1101/2023.11.10.566609v2 on bioRxiv
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    Referee #4

    Evidence, reproducibility and clarity

    In this study Ermanoska and Rodal explored how the presynaptic actomyosin and its subcellular organization and function are assembled and how they respond to mechanical forces. In particular, the authors describe a new type of actin assembly that extends as a continuum through the Drosophila NMJ: this linear actin assembly is in part co-localized with NMII and with Tropomyosin, which led the authors to hypothesize that it may have contractile properties. They follow with knock down (KD) experiments of NMII in motor neurons and show that this KD changes linear actin and also reduces postsynaptic NMII and Integrin receptor levels (pre- and post-synaptically). This data suggests an intricate trans-synaptic molecular interplay between motor neurons and the muscle. Finally, in Figure 6 the authors manipulate axonal mechanical tension through the cutting or not cutting of the nerve bundle and argue that mechanical tension is also required to maintain this type of linear actin core. Altogether, this manuscript describes a potentially very interesting phenomenon whereby mechanical forces contribute to neuronal structure, namely through the control of actin types of assembly and provides some data supporting that actin/NMII/Integrins interact trans-synaptically to transmit force information between cells.

    However, in its current format this study is a bit preliminary and mechanistically incomplete. The data regarding the description of 2 distinct types of actin assemblies, with distinct half-lives and stability is convincing, and well-documented but the remainder of the manuscript is more preliminary and not fully sustained by the data presented. The data regarding mechanical forces is particularly unprecise, but it can potentially unveil a novel mechanism that (at least in part) explains how force and biochemical signaling are integrated by neurons. In sum, this manuscript describes an interesting topic but the current version can be significantly improved with additional experiments and/or controls.

    Below are my specific comments. If addressed, this manuscript should be published as it significantly adds to the emerging field of mechanobiology and intercellular communication. It provides a new way to look at the effect of mechanical forces in the context of synaptic biology.

    Major comments and suggestions for experiments:

    • In the images presented on Fig. 2A and 2B, both Arp2-3-GFP and Dia-GFP seem to co-localize with the filamentous F-actin signal, and the authors state this. However, the Pearson correlation is weak, leading the authors to "remove" this claim. On the contrary, the Tm signal is said to have a strong Pearson Correlation. However, looking at the images, it is very hard to understand why the signals are not correlated. Can the authors explain how they quantified the correlation? If Arp2-3-GFP and Dia-GFP are not enriched on linear F-actin, the chosen images are not appropriate.Alternatively, can the authors find a better way to assess colocalization? % of puncta colocalized? Also, I suggest that the quantification of these data, which is currently on Fig. S3 to be moved to the main figure 2.
    • Also on Figure 2D, the Lifeact::Halo is a lot smoother than on the other panels with the same marker, and is very much alike the QmN-Tm signal, raising the possibility of a bleed-through artifact. Given that the authors have an antibody against Tm1, can they use it on larvae that express Lifeact::Halo (without QmN-Tm1) to confirm the degree of co-localization (which based on Figure 2E appears as the authors claim, but that is not very convincing on Fig.2D, where it looks like there may be some bleed-through of the channels).
    • In figure 3, for consistency, can the authors use Lifeact in zip KD rather than GMA? Or is there a specific reason for this change relative to Fig. 1 and 2? Alternatively, it would be important to show that GMA and Lifeact have similar expression patterns, by co-expressing them simultaneously.
    • Figures 2 and 3 raise the idea that there are contractile actin fibers, and this is an important message of this paper. Therefore, it would help to have additional data regarding the manipulation of NMII. Namely, 1) whether expressing RNAi against Sqh gives rise to the same effects as the KD of Zip, and 2) what is the effect of expressing UAS-Sqh CA (phosphomimetic) and UAS-Sqh DN (non phosphorylatable) on linear actin and on the levels of postsynaptic NMII, and pre- and post-synaptic Integrin receptor levels.
    • The idea of NMII neuronal KD influencing postsynaptic NMII levels is rather intriguing and potentially very interesting. Is this interaction reciprocal? What happens if Zip is KD in the muscle? Does it influence presynaptic NMII levels? Same comment for Integrin staining. Also, can the authors comment on how they envision that NMII KD can lead to a generalized reduction in the whole muscle? NMII and Integrin should be quantified in non-synaptic and synaptic areas of the muscle.
    • The difference in intensity of NMII and Integrins is quite striking and meaningful in terms of trans-synaptic signaling. To validate the quantifications shown in Figures 4 and 5, it is critical to be confident that the larvae analyzed are both time and size matched. Because the authors don't state it clearly, it is a formal possibility that the developmental timing is slightly different between controls and KDs, which could lead to lower levels of NMII and Integrins due to timing rather than manipulation or genotype. If this is the case, the two situations (time and size matching) should be analyzed for post-synaptic reductions of NMII and Integrins. To further confirm a direct effect of NMII KD leading to pre- and post-synaptic alterations of NMII and Integrins, it would be important to use a neuronal line that is expressed in a subset of motor neurons and compare with non-expressing NMJs in the same larvae. This would remove possible effects of the developmental timing. Additionally, since every marker analyzed is reduced, it would be important to find a marker that is unaltered by the KD of Zip (FasII?). Without these controls/extra experiments, the claims regarding NMII and Integrin reduction are not well supported.
    • Figure 6: in this figure the authors cut the nerve and then measure actin intensity, and types of actin assemblies. This data is used to conclude that axonal severing impacts mechanical properties of axons and changes actin distribution and types of assemblies. Even though the concept is novel and interesting, the data is not sufficient for the claims. Ideally, it would be important to be able to control and quantify the stretch force applied and the level that is required to promote the distinct types of actin structure. I do understand that these experiments may be difficult to perform, and may require methodologies that are not standard. However, there are ways to improve this data. For example, since these measurements of actin levels and distribution are performed live, it would be important to do a time-lapse movie to understand how linear actin is lost and puncta of actin increase, followed by a quantification of these parameters.

    Even though it is hard to provide a "force number", it is relatively simple to repeat the experiment from Figure 6 in conditions of cut and uncut nerve, but adding a stretched nerve condition. Does stretch promote linear actin? To perform this experiment, the authors can pull the brain and its nerves up and glue it in a way that the nerve bundles are connected to the NMJ but are more stretched than in the dissected "loose" condition. Additionally, the authors should analyze how manipulation of actin polymerization (LatA and JASPA) impact this process. Finally, since the authors show in Figures 4 and 5 that manipulations that result in the decrease of linear actin leads to reductions of Integrins and NMII, they should assess if changing the mechanical tension of the nerve also impacts these signaling pathways.

    • Perhaps a bit out of scope, but very much related: what happens to actin structure after muscle contraction? In other words, does mechanical pressure at the NMJ also alter actin?

    Minor comments:

    • In all Figures, it is not stated from how many independent experiments/crosses are the data derived from. In most experiments, the number of larvae analyzed is on the low end.
    • In Figure 3 and Figure S5, in the zip KD (at least by eye) bouton size looks increased. Is there a difference? Since it looks obvious by eye, can the authors quantify this morphological feature, that can also be related with an actomyosin cortex?
    • Can the authors specify that the control UAS-BL35785 is and RNAi against mCherry (in the Tables and perhaps also in the legend)?
    • In the discussion, the authors state that they "We took advantage of the Drosophila model and targeted NMII directly by neuronal depletion of both the heavy chain and light chain of NMII. Interestingly, we observed major perturbations of presynaptic actin subpopulations, including of the linear presynaptic actin core." Unless I am missing some Figure, I could not find this data regarding Sqh. The KD of Sqh appears only in Supp Figure 4, to validate the efficacy of KD and not actin. This should be corrected.

    Methods:

    • Can the authors say if the crosses were performed in vials or cages? This can significantly change some NMJ parameters.
    • Extra information regarding the mounting of the larvae for live imaging can be provided: if the larvae is not fixed, how do the authors control the positioning in the drop of HL3.1? How is the stretching/non-stretching of the nerve controlled for? Or are the larvae glue on the side with the double-sided sticky tape? These details can be provided to assure reproducibility by other labs.
    • If I understood correctly, in the LatA experiment, the larvae are imaged in the absence of LatA. This is not clear in the results section and should be corrected.
    • Please provide more details on how were the correlations performed?

    Significance

    This study describes the existence of an new actin assembly, linear actin, that extends through the Drosophila larval NMJ. To my knowledge this is reported for first time and has functional implications, since the authors hypothesize that this structure has contractile properties. This study also proposes that mechanical forces can directly be sensed by actin, which modifies its structure and alters signaling molecules at the synapse, namely through transsynaptic signaling, via Integrins. Altogether, the idea represents a novel concept, with an attempt to provide some mechanistic detail (even though it lacks data to support some of the hypothesis).

    This study is of interest to both specialized and broad audiences, interested in basic research.

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    Referee #3

    Evidence, reproducibility and clarity

    The advent of super-resolution microscopy has dramatically increased our understanding of the organization and function of the cytoskeleton in neurons. However, there are still areas which remain poorly understood, particularly in neuronal subtypes that are not conventional models for studying the neuronal cytoskeleton. Here Ermanoska and Rodal use super-resolution microscopy and improved probes for imaging actin in Drosophila motor neurons and have identified a novel linear actin structure in the presynaptic terminal of motor neurons. This linear structure appears to be regulated by non-muscle myosin 2 and is important in maintaining the integrity of the neuromuscular connection. For example, the authors show that depleting NM2 in the neurons alters the amount of linear F-actin and the distribution of integrins at the presynaptic terminal. Additionally, performing an axotomy also reduces these linear structures at the nerve terminal, presumably due to decreased tension along the neuron.

    Since this is a review of a preprint, I will limit my assessment of the manuscript to what I feel are the major issues in the hopes that it will be helpful to the authors in reworking the manuscript for submission. Most of these points could be addressed in multiple ways.

    Major issues and outstanding questions:

    1. Axonal actin bundles have been previously identified, though that would not have been clear from reading this paper. The work of Ganguly et. al, JCB 2015; Chakrabarty et al, JCB 2019; Phillips et al., J Neurosci Methods; Gallo J Cell Sci 2006; Brown and Bridgeman Dev. Neurobiol 2009; Orlava et al. Dev. Neurobiol 2007; and Ketshek et al eLife 2021 should be cited and discussed in the context of this work. Interestingly, many of the linear bundles of actin filaments described above are associated with NM2-dependent axonal retraction. The works should be cited and discussed in the context of the results found in this manuscript.
    2. Are there similar bundles along the axons of these motor neurons, or do they only occur at the presynaptic terminal? Or does the type of imaging and model system being used only allow for these structures to be visualized at the presynaptic terminal?
    3. The term "Molecular composition of linear actin structures" is being overused here- you are only showing the colocalization of tropomyosin 1.
    4. If Tm1 is important for these structures, why are they still present when it is deleted? I do not see the quantification of linear actin when Tm1 is depleted. Additionally, when integrin redistribution is being measured in Sup. Fig 6, I do not see the Tm1 depleted data despite Tm1 being in the title of the figure.
    5. Is there an increase in activated NM2 at the presynaptic terminal? What happens if you increase NM2 activity in these neurons?
    6. There is a depletion of NM2 particles in the postsynaptic terminal when NM2 is being depleted in only the neurons- but is NM2 expression being affected in the muscle cells or only localization of puncta to the nerve terminals?
    7. What is the functional consequence when linear actin structures are depleted- Denervation? Decreased synaptic activity? Anything?
    8. It would really help to strengthen the conclusions of this paper if NM2 could be locally and acutely activated or inactivated at the nerve terminal. Nearly all the phenotypes observed are due to global perturbations that may have broad consequences.
    9. Are these structures present at the presynaptic nerve terminal in other species? If not, or if you do not want to look into it, then it might be more appropriate to add "in Drosophila" to the title.

    Significance

    This manuscript presents an exciting concept that will be of high interest to cellular neuroscientists and cytoskeletal biologists. There are also interesting implications that could be made with aging and neurodegenerative diseases of the neuromuscular system. The manuscript is well written and contains rigorous experimentation and analysis of the data. My main issue with it, however, is that the conclusions seem preliminary and are heavily reliant on correlation. Additionally, there is a complete lack of discussion of similar structures that have been seen in axons. Finally, all of the data is from one cell type from a single species, which limits how broadly the results can be interpreted and whether this data has potential relevance to human aging/disease, which would help it reach a larger audience. Basically, I am confident that the data that is presented is correct, though it is potentially being overinterpreted when being put into a broader context.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: In this study, Drosophila larval NMJs were used to investigate the very interesting and innovative hypothesis that actomyosin-mediated contractility generates and responds to cellular forces at the neuron-muscle interface. In summary, the authors identified a new presynaptic actomyosin subpopulation that transmits signals to adjacent muscle tissue that together with with integrin receptors governs the mechanobiology of the neuromuscular junction.

    While this study presents exciting evidence supporting the existence of a cable-like actomyosin structure traversing the NMJ, some of the conclusions are not fully supported by the data provided. It is unclear how this actomyosin arrangement differs (or not) from other longitudinal myosin arrangements found in the axon shaft. In this respect, it would be informative to provide images of the axon shaft to further verify the possible presynaptic specificity of this actomyosin arrangement, and check whether alternatively it might exist as a continuum of actin cables already present in the axon shaft.

    The data presented in Figure 2F is insufficient to claim that a presynaptic actomyosin core exists. As it is, the myosin puncta shown do not definitely support that such a structure exists. Alternative approaches such as using fluorescent NMII fusions that allow visualizing simultaneously the N- and C-terminal domains of the NMII heavy chain could be used.

    Claims on the effect of the neuronal actomyosin assemblies on tension, in the absence of experiments directly assessing tension, should be down toned.

    Also, the data provided in the axotomy experiments is not sufficient to claim that axonal severing is sensed specifically at the presynaptic terminal in a similar manner to neuronal NMII depletion. Axotomy is certainly followed by degeneration and dismantling of different axonal cytoskeleton compartments including the formation of altered actin arrangements, including those of the presynaptic terminal.

    Significance

    This is a very interesting study that raises a novel hypothesis on how neuronal mechanobiology is governed. If complemented with additional experiments further supporting the existence of a specific actomyosin arrangement in presynaptic terminals, this study will certainly be of high significance to the field and of broad interest to readers that are not experts on the topic.

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    Referee #1

    Evidence, reproducibility and clarity

    In this study, Ermanoska and Rodal describe the features of a recently described (by the same group) presynaptic entity in the NMJ. The authors find evidence of diverse types of actin assemblies along the presynaptic contact, patches, and cables (similar to structures observed during fission yeast division). Among these proteins, NMII (Sqh) seems prominently featured. Zip mutations apparently alter the distribution of the actin, albeit modestly, and also affect integrin patching at the synapse. Finally, the authors provide evidence that mechanical severing induces specific actin remodeling.

    The study is provocative, but some of the conclusions of the study are quite evident and predictable. Also, the localization of the proteins at presynaptic cables is not as clear as the authors describe them. Finally, the effects of NMII depletion using siRNA are compounded by possible off-targets effects that the authors shrewdly attribute to presynaptic-specific phenotypes. Proof of this is quite weak and it seems likely that some neuron-specific promoters are leaking beyond neurons.

    Major issues:

    • The authors have made a large effort to characterize the presynaptic actin structures in as much detail as possible, but this reviewer is apprehensive regarding the validity of the observations made in the presence of highly perturbing probes. It is well-known in the field that most actin-binding probes, including moesin-actin BD, Lifeact, utrophin, etc., have no perturbing effects... except in neurons. In their previous publication (eLife 2017), the authors used GFP-actin (which display binding kinetic alterations), MA and Lifeact, and got away with it. They never stained with phalloidin, which is the gold standard for unperturbed F-actin visualization. Given the level of structural detail the authors are getting into, they need to address the visualization of these structures in a totally unperturbed manner.
    • Sqh:GFP does not really localize in the structures, but everywhere (Fig. 2F). Again, Sqh:GFP is a notoriously flaky probe (DOI: 10.1002/cm.21212) that makes this reviewer nervous in the absence of additional validation, which in this case may take the form of HA/myc/FLAG-tagging (which require staining but does not interfere with Zip:Sqh binding) or endogenous staining, particularly with phospho-specific antibodies (for use in Drosophila samples, see for example DOI: 10.1038/emboj.2010.338).
    • What is the actual efficiency of NMII depletion? This is a stubborn molecule difficult to deplete efficiently in most systems.
    • The authors observed that NMII depletion driven by RNAi under a neuronal specific promoter also reduces NMII expression in the post-synaptic region and the muscle. The authors claim that this is specific and not leaky by examining NMII expression in the absence of C155-Gal4. To the extent of this reviewer's knowledge, this is thus based on the specificity of C155. However, it has been well documented and explicitly stated that Drosophila enhancer-Gal4 lines show ectopic expression during development (paper by this title, using C155-Gal4 among other promoters, DOI: 10.1098/rsos.170039). Those authors observed expression in wing cells, for example, which casts severe doubt on this particular conclusion.
    • What would be the effect of severing in NMII-depleted presynaptic assemblies?

    Referees cross-commenting

    I concur with the comments of my esteemed colleagues. Still, I am concerned regarding the use of the C155-Gal4 promoter and its effects outside of neurons. The conclusion that that NMII depletion driven by RNAi under a neuronal specific promoter also reduces NMII expression in the post-synaptic region and the muscle is potentially the most striking finding of the paper, but the fact that this promoter (which is potentially leaky) is used dampens my enthusiasm. Also, the use of the actin probes is a problem, and one I don't see fixed by the fact they published a previous paper before using them. Maybe the reviewers then had less or no experience with these probes. I have in the past, and I cannot let this slide

    Significance

    As described in the previous section, the study has several built-in limitations that dampen this reviewer's enthusiasm for the overall story, including the limitations of the molecular tools used, which are quite-artifact prone (this reviewer has plenty of firsthand experience with all these tools in mammalian models, and has suffered some of them to become big, months-consuming artifacts). Also, the authors use fly lines that either are leaky; or they elect not to explore the most interesting piece of data in the paper, which is the transsynaptic effect on NMII expression. This reviewer suspects that the authors have not pursued this vigorously because they have their own suspicions in this regard.

    If properly carried out, this study would have filled an important gap, since most existing studies have so far focused on the post-synaptic region, hence it'd be important to find out precisely what is happening on the other side. But this study does not clarify this.

    The audience would have been mainly cell biologists, cellular neurobiologists and "fly people", with some transversal interest from the budding mechanobiology community. But the story is quite flawed, beyond revision given the approaches used (and trusted) by the authors. I cannot recommend publication of this manuscript if the issues raised here are not addressed.

  7. Version published to 10.1101/2023.11.10.566609v1 on bioRxiv