Microtubules under mechanical pressure can breach dense actin networks

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Abstract

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    Major comments
    In the paper "Microtubules under mechanical pressure can breach dense actin networks", the authors showed clear evidence that pressure plays an important role in microtubule breaching into dense actin networks using elegant in vitro reconstitution assays. They have argued that the pressure results from polymerization force of microtubules, which builds up when microtubules are immobilized in the opposite end of breaching, by the means of actin microtubule crosslinking factor Tau.

    Authors answer:

    We thank the reviewer for his/her positive comments on our manuscript.

    It would definitely be interesting to see lack of breaching in the presence of crosslinking deficient Tau construct in order to rule out the off -target effect of Tau on microtubule and actin architecture which may possibly facilitate breaching.

    Authors answer:

    This is an interesting suggestion. Unfortunately, we do not have in hand such crosslinking deficient Tau construct. However, please note that we showed two independent ways to demonstrate the role of pressure. One is indeed by crosslinking microtubule to actin bundle with Tau, but the other is by blocking the two opposite ends of microtubules with two dense actin networks. So, we think our conclusion about the role of pressure is solid.

    The authors have also observed microtubule breaching into dense actin networks in living cells. However, in Figure 1C, better cell/ image processing might have been chosen to increase the visibility of actin structures that microtubules encounter on their way to breaching. In Figure S1D, for example, the similar actin structures in lamellipodia are very nicely visible.

    Authors answer:

    We apologize but we don’t understand reviewer’s comment. In figure 1C images of actin networks are shown in black and white and are more visible than in figure S1D where they are shown in magenta and overlaid with microtubules. In any case, we increased the contrast of images to make fine actin structures at the cell edge clearer.

    It is also interesting that on Figure 6A, actin bundles look different than the rest of the figures on the paper. It almost looks like actin bundles become branched, whereas in the other Figures actin bundles are either singular or two-three bundles joined together at the point very close to the edge of micropatterned lipid bilayer.

    Authors answer:

    This is correct. In this experiment several bundles co-aligned. As mentioned by the reviewer this could also be visible in other conditions without Tau (such as in Figure 4E), and, as shown below, this structure of bundle was not visible in all fields we looked at. So we don’t think this structure is responsible for the changes we measured in the ability of microtubules to penetrate the actin network in the presence of Tau.

    Minor comments
    In the legend of Figure 4E, it should be written "white arrow" instead of "yellow arrow".
    In the Results section "crosslinking between microtubules and actin bundles increase piercing frequency", in the sentence number 7, it should be written "backwards" instead of "reaward".

    Authors answer: We modified the text and legend according to the reviewer suggestions.

    Reviewer #1 (Significance):

    The experimental setup of the paper is quite significant in the field, given the difficulty of observing dynamics of dense cytoskeletal structures in living cells. Moreover, the paper gives insight into how microtubule behavior can vary depending on different morphological states of actin network.

    Authors answer: We thank the reviewer for his/her overall very positive feedback on our manuscript.

    Reviewer #2 (Evidence, reproducibility and clarity):

    The authors developed a novel in vitro system to investigate the interaction of dynamic microtubules with the F-actin network. While this system does produce some interesting results, it is unclear how exactly this replicates or explains what might happen near a cell's leading edge. There is a limited characterization of the produced F-actin networks. For example, it is unclear to what extent the F-actin networks are similar or different to cell lamellipodial networks. What is the density / expected mesh size of these networks and could that be varied / manipulated? The bottomline observation that microtubules can grow into F-actin networks if they have nowhere else to go does not seem particularly ground-breaking, and the discussion is very shallow. Overall writing could be improved; there are lots of typos and grammatical inconsistencies. The second paragraph of the introduction is a bit convoluted.

    Authors answer:

    We thank the reviewer for his/her comments. Figure 1 was used to illustrate the behavior of microtubules encountering actin networks in cells and the fact that they struggle to penetrate actin network. This is only a way to argue that the penetration of actin network is a relevant question, that cannot be easily addressed in cells. However, it is correct that our in vitro systems, as it is the case for all in vitro reconstituted systems, cannot tend exactly to reproduce a lamellipodial cellular network. But it offers a better way to modulate actin network architecture. We have used in vitro systems to characterize the different behavior of microtubules when they encounter dense actin networks in different conditions, guided or not by actin bundles, constraint or not at the two ends.

    The observation that microtubule can penetrate actin network when pressurized might not be “ground breaking”, still it contradicts previous works showing that microtubule under pressure tend to depolymerize (Janson et al, J Cell Biol, 2003), which would obviously prevent them from penetrating actin networks. So, our conclusion was somehow unexpected.

    We found important to discuss the fact that although the microtubule polymerizing forces is sufficient to breach dense actin network, it must be counteracted by another mechanism immobilizing microtubules. This means that in cells, expression level of actin-microtubule crosslinker modulate the penetration of microtubule into the lamellipodium.

    However, we agree that the second paragraph of the introduction is not absolutely necessary and removed it.

    Specific comments:

    Fig. 1 seems a bit anecdotal. The authors revisit an observation that has been made before. I can see how it is used as rationale for the in vitro system, but not sure that this adds much to the overall story. Clearly different cell types are different, but without some sort of quantification this remains meaningless. It should also be noted in the discussion maybe that there are large differences between cells in 2D and 3D. Microtubules much more frequently grow to the cell edge compared with 2D (see Akhmanova SLAIN2 paper from some years ago).

    Authors answer:

    We agree with these comments. Indeed, Figure 1 is used only as an illustration of the behavior of microtubules encountering actin network in cells. As the reviewer said, microtubule penetration and actin architectures will both vary a lot from one cell type to another. So we believe that quantification for these particular cases will not extend the illustrative purpose of this figure where it is already clear that some microtubules can penetrate and other can’t.

    Fig. 2: While Arp2/3 certainly promotes branched F-actin networks, from the data provided it is not clear to me to what extent the produced F-actin networks replicate F-actin organization at the cell edge. If this a the point the authors are trying to make, the ultrastructure of their in vitro networks needs some additional characterization. As far as it is possible to discern from the data provided, the F-actin meshwork on the stripes in E looks pretty much identical in both panels (and not really like a dendritic network that in a cell also would have a certain polarity with barbed ends facing out), and the bundles on the left don't look like anything that normally occurs in a cell.

    Authors answer:

    We also agree with these comments. The networks we assembled are not lamellipodial-like networks, there are branched network of various densities, with or without bundles. It is true that bundles of filaments do not grow out of lamellipodial network in cells. However, bundles of aligned and linear filaments exist in cells, in the form of radial fibers or transverse arcs, along which microtubule tend to align. And these structures might guide microtubules toward cell protrusions, as it is the case in growth cone for example.

    Fig. 4 It is unclear what is going on here. Given that without F-actin bundles, polymerizing microtubules are freely moving around, it does not come as a surprise that they would never penetrate the F-actin network because as the authors correctly state the growing end will push back from the barrier. So, then why do they sometimes penetrate when bundles are present? In 4A it appears that microtubule growth into f-actin only happens once the microtubule minus ends gets stuck between F-actin bundles on the other side. 4D is the same as 4A; so that makes me think this really does not occur that often. Does the microtubule plus end only penetrate the F-actin meshwork when the minus end gets stuck on the other side? This seems important and also means microtubule penetration may not have anything to do with the F-actin network architecture at the plus end. This needs to be quantified.

    Authors answer:

    This is perfectly correct. In figure 4 the two actin networks are distant, and the microtubules only rarely penetrate them because they are rarely in contact with them at both ends. This occurs only when bundles orient microtubules perpendicular to the edges of the actin network, since in this configuration the distance between the two actin networks is shorter. Hence our motivation to bring actin networks closer to each other in figure 5.

    Fig. 5 I guess that sort of solves my confusion with Fig. 4. The quantification graphs in 5B and 5C are flipped with respect to the figure legend (?).

    Authors answer:

    Indeed, in this figure we distinguished the role of pressure (when both microtubule ends are in contact with actin networks) and the role of alignment with actin bundles. And found that the presence of bundles is useless and that only pressure matters.

    I understand the rationale for not considering microtubules that grow at a shallow angle, but there does not seem to be that much of a difference between 5B and 5D. Wouldn't a better quantification simply compare microtubules that contact F-actin at both ends compared with microtubules were the minus end is free. In this case, I would expect a very large difference in penetration.

    Authors answer:

    This is also correct. The difference is so important that when one end is free the microtubule never penetrate. We mention it in the text but did not plot these data. This is why we measured only microtubule with both ends contacting an actin network and did not consider the one at shallow angles.

    We added the illustration of the condition with short distance and actin bundles (shown below) to make this more clear in the figure.

    The small difference between 5B and 5D shows that by eliminating those microtubules there is no more difference between the conditions with or without bundles. And thus that their contribution in favoring microtubule penetration was to favor optimal orientation to get pressurized at the two ends rather than offering a sort of favorable network organization at their base. However, we agree with the reviewer that the absence of difference between the two populations, with or without actin bundle, when considering only microtubule interacting with actin at angles higher than 30° is not quite striking. We tested all angles (see below) and found that actually the absence of difference is more obvious when considering microtubules interacting with more than 60°. And the analysis of angle distribution, now reported in Figure 5D, showed that in both conditions most microtubules interact with more than 60°, so we only exclude few outliers by considering those that interact with more than 60°. So we changed the presentation of our data in Figure 5C by changing the threshold from 30 to 60°.

    Do microtubules under pressure ever bend/buckle in this in vitro situation. As the authors state, in cells, that happens frequently. This difference is interesting. Why?

    In vitro microtubules buckle homogeneously between their two ends. These long buckling wavelengths are not very spectacular. In cells, microtubules are crosslinked to actin filaments or other structures over shorter distances (see quantification below). This leads to buckling with shorter wavelength, which is more striking.

    It is customary to refer to polymerized actin as F-actin.

    The supplementary videos are not referenced in the manuscript.

    Authors answer:

    We apologize and have now referenced the supplementary video in the manuscript.

    Reviewer #2 (Significance):

    The manuscript describes results from a novel assay to study interactions between F-actin networks and dynamic microtubules in vitro. While of interest to a specialized audience, the overall finding that microtubules can grow into an F-actin meshwork is somewhat incremental especially because of the limited characterization of the F-actin networks used. It remains unclear to what extent this is relevant to a physiological context in cells.

    My field of expertise is related to cytoskeleton dynamics and quantitative microscopy in live cells.

    Authors answer:

    Although intuitive, the demonstration that the density of actin network can prevent microtubule penetration is novel. More importantly, the demonstration that anchoring of microtubule is sufficient to increase the pressure to such a point that microtubule can then penetrate those networks is also novel and significant to appreciate when and how they do so in cells.

    Reviewer #3 (Evidence, reproducibility and clarity):

    In this paper, the authors present an in vitro assay designed to explore the dynamic interaction between growing microtubules and pre-existing actin networks. Notably, the presence of linear actin bundles facilitated the movement of polymerizing microtubules along actin filaments. When microtubules were immobilized to two spatially separated actin networks, they exhibited the ability to breach and penetrate dense actin meshworks. This penetration was attributed to the mechanical pressure generated by microtubule polymerization. The authors tested tau as a microtubule-actin crosslinking protein in this process and found that tau promoted microtubule penetration into dense actin meshwork. Although the findings in this paper are potentially significant, the work is still in its preliminary stage and the scope is limited.

    Authors answer:

    We thank the reviewer to summarize properly the main findings of our manuscript.

    1. The authors observed that the inclusion of tau, a microtubule-associated protein known for its role in promoting microtubule polymerization, significantly facilitated microtubule penetration into dense actin meshworks. This enhancement is likely attributed to tau's ability to promote microtubule polymerization, generating stronger forces within the microtubules that enable them to breach the actin meshworks. To validate the involvement of the crosslinking function in the process, the authors should explore the effects of other microtubule-actin crosslinking proteins in their assay.

    Authors answer:

    We thank the reviewer for this interesting suggestion regarding the role of Tau in our experiments. To address this comment, we have analyzed the rate of growth in our experiments in presence and absence of Tau (see quantification below). We found that the construction of Tau we used reduced microtubule growth rate. Therefore, we believe that microtubule growth was not responsible for the improved penetration of microtubule in dense actin networks in our assay, and that it was rather the crosslinking ability of Tau that played a significant role.

    1. The paper highlights the importance of anchoring both ends of microtubules to two adjacent actin networks for successful penetration into the actin meshworks. However, the precise mechanisms by which these microtubule ends are anchored to actin filaments are not elaborated upon. Providing detailed insights into this anchoring process would enhance the readers' comprehension of the experimental setup and its relevance to the observed results.

    Authors answer:

    We apologize for this lack of clarity. We don’t think that microtubule ends are “anchored” to the actin network. They are simply embedded into it. This embedding prevents them from moving rearward and thus lead to pressure increase as they polymerize.

    1. Additional information on the experimental methods is warranted to improve the reproducibility and clarity of the study. Specifically, the authors should elucidate the process through which nucleation-promoting factors were grafted onto lipid bilayers. This detail is crucial for researchers seeking to replicate or build upon the study's findings.

    Authors answer:

    We apologize for this lack of clarity. There was indeed an error in our description of SUV preparation with lipid-biotin. We have now revised our material and method section. In particular we have described more accurately the various steps we used to micropattern WA-streptavidin onto lipid-biotin.

    1. In Fig. 5D, the authors observed no significant difference in the breaching probability between microtubules that contacted the actin meshwork at an angle higher than 30°, with or without actin bundles. To ensure a better comparison, it is advisable to focus on quantifying the microtubules that are contacting two actin meshworks at both ends (the immobilized microtubules), as they would have similar probabilities of being pressurized by their growth. Moreover, further justification is required to explain the choice of 30° as the threshold angle and its significance in the context of microtubule behavior.

    Authors answer:

    We thank the reviewer for this comment. We apologize for the confusion. The quantification we made is precisely the one described by the reviewer. We made this more clear by adding further illustration of the two conditions and the measurement made.

    1. Fig. 5C appears to depict the "Distribution of the angle of the interaction of microtubules in the presence (10nM of Arp2/3 complex) or absence (100 nM of Arp2/3 complex) of actin bundles" instead of the "proportion of microtubules piercing the branched actin network." The alphabet labels in the figure should be updated accordingly. Additionally, the authors should clarify whether a comparison was conducted between the means of the angles in the two conditions and whether any observed differences were statistically significant.

    Authors answer:

    We apologize for this confusion. We updated the figure legend in which 5C and 5D were inverted.

    1. Investigating the potential significant difference in the mean interaction angles between the absence and presence of actin bundles would be intriguing. The presence of actin bundles might indeed influence the interaction angle or contact position, potentially increasing penetration frequency. This insight would further enrich the findings and provide valuable context for understanding the interplay between microtubules and actin networks.

    Authors answer:

    We apologize for this confusion. We now report the statistical difference. And indeed, it accounts for the difference it the penetration frequency, as shown by the absence of difference when we consider only microtubules that are more or less perpendicular to the network. This is indeed one of the most significant conclusion of our work. We added some schematics to make this clearer.

    1. More comprehensive information about the statistical analyses should be provided. This'd be important for the validity and reliability of the study's conclusions.

    Authors answer:

    We apologize for this lack of clarity. The statistical analysis we performed were not described in the Materials and Methods section but in each figure legend.

    Reviewer #3 (Significance):

    The work represents an advance in understanding the mechanism by which microtubules navigate dense actin meshworks.

    Authors answer:

    We thank the reviewer for this positive evaluation of our work.

  2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    In this paper, the authors present an in vitro assay designed to explore the dynamic interaction between growing microtubules and pre-existing actin networks. Notably, the presence of linear actin bundles facilitated the movement of polymerizing microtubules along actin filaments. When microtubules were immobilized to two spatially separated actin networks, they exhibited the ability to breach and penetrate dense actin meshworks. This penetration was attributed to the mechanical pressure generated by microtubule polymerization. The authors tested tau as a microtubule-actin crosslinking protein in this process and found that tau promoted microtubule penetration into dense actin meshwork. Although the findings in this paper are potentially significant, the work is still in its preliminary stage and the scope is limited.

    1. The authors observed that the inclusion of tau, a microtubule-associated protein known for its role in promoting microtubule polymerization, significantly facilitated microtubule penetration into dense actin meshworks. This enhancement is likely attributed to tau's ability to promote microtubule polymerization, generating stronger forces within the microtubules that enable them to breach the actin meshworks. To validate the involvement of the crosslinking function in the process, the authors should explore the effects of other microtubule-actin crosslinking proteins in their assay.
    2. The paper highlights the importance of anchoring both ends of microtubules to two adjacent actin networks for successful penetration into the actin meshworks. However, the precise mechanisms by which these microtubule ends are anchored to actin filaments are not elaborated upon. Providing detailed insights into this anchoring process would enhance the readers' comprehension of the experimental setup and its relevance to the observed results.
    3. Additional information on the experimental methods is warranted to improve the reproducibility and clarity of the study. Specifically, the authors should elucidate the process through which nucleation-promoting factors were grafted onto lipid bilayers. This detail is crucial for researchers seeking to replicate or build upon the study's findings.
    4. In Fig. 5D, the authors observed no significant difference in the breaching probability between microtubules that contacted the actin meshwork at an angle higher than 30{degree sign}, with or without actin bundles. To ensure a better comparison, it is advisable to focus on quantifying the microtubules that are contacting two actin meshworks at both ends (the immobilized microtubules), as they would have similar probabilities of being pressurized by their growth. Moreover, further justification is required to explain the choice of 30{degree sign} as the threshold angle and its significance in the context of microtubule behavior.
    5. Fig. 5C appears to depict the "Distribution of the angle of the interaction of microtubules in the presence (10nM of Arp2/3 complex) or absence (100 nM of Arp2/3 complex) of actin bundles" instead of the "proportion of microtubules piercing the branched actin network." The alphabet labels in the figure should be updated accordingly. Additionally, the authors should clarify whether a comparison was conducted between the means of the angles in the two conditions and whether any observed differences were statistically significant.
    6. Investigating the potential significant difference in the mean interaction angles between the absence and presence of actin bundles would be intriguing. The presence of actin bundles might indeed influence the interaction angle or contact position, potentially increasing penetration frequency. This insight would further enrich the findings and provide valuable context for understanding the interplay between microtubules and actin networks.
    7. More comprehensive information about the statistical analyses should be provided. This'd be important for the validity and reliability of the study's conclusions.

    Significance

    The work represents an advance in understanding the mechanism by which microtubules navigate dense actin meshworks.

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #2

    Evidence, reproducibility and clarity

    The authors developed a novel in vitro system to investigate the interaction of dynamic microtubules with the F-actin network. While this system does produce some interesting results, it is unclear how exactly this replicates or explains what might happen near a cell's leading edge. There is a limited characterization of the produced F-actin networks. For example, it is unclear to what extent the F-actin networks are similar or different to cell lamellipodial networks. What is the density / expected mesh size of these networks and could that be varied / manipulated? The bottomline observation that microtubules can grow into F-actin networks if they have nowhere else to go does not seem particularly ground-breaking, and the discussion is very shallow. Overall writing could be improved; there are lots of typos and grammatical inconsistencies. The second paragraph of the introduction is a bit convoluted.

    Specific comments:

    Fig. 1 seems a bit anecdotal. The authors revisit an observation that has been made before. I can see how it is used as rationale for the in vitro system, but not sure that this adds much to the overall story. Clearly different cell types are different, but without some sort of quantification this remains meaningless. It should also be noted in the discussion maybe that there are large differences between cells in 2D and 3D. Microtubules much more frequently grow to the cell edge compared with 2D (see Akhmanova SLAIN2 paper from some years ago).

    Fig. 2: While Arp2/3 certainly promotes branched F-actin networks, from the data provided it is not clear to me to what extent the produced F-actin networks replicate F-actin organization at the cell edge. If this a the point the authors are trying to make, the ultrastructure of their in vitro networks needs some additional characterization. As far as it is possible to discern from the data provided, the F-actin meshwork on the stripes in E looks pretty much identical in both panels (and not really like a dendritic network that in a cell also would have a certain polarity with barbed ends facing out), and the bundles on the left don't look like anything that normally occurs in a cell.

    Fig. 4 It is unclear what is going on here. Given that without F-actin bundles, polymerizing microtubules are freely moving around, it does not come as a surprise that they would never penetrate the F-actin network because as the authors correctly state the growing end will push back from the barrier. So, then why do they sometimes penetrate when bundles are present? In 4A it appears that microtubule growth into f-actin only happens once the microtubule minus ends gets stuck between F-actin bundles on the other side. 4D is the same as 4A; so that makes me think this really does not occur that often. Does the microtubule plus end only penetrate the F-actin meshwork when the minus end gets stuck on the other side? This seems important and also means microtubule penetration may not have anything to do with the F-actin network architecture at the plus end. This needs to be quantified.

    Fig. 5 I guess that sort of solves my confusion with Fig. 4. The quantification graphs in 5B and 5C are flipped with respect to the figure legend (?). I understand the rationale for not considering microtubules that grow at a shallow angle, but there does not seem to be that much of a difference between 5B and 5D. Wouldn't a better quantification simply compare microtubules that contact F-actin at both ends compared with microtubules were the minus end is free. In this case, I would expect a very large difference in penetration. Do microtubules under pressure ever bend/buckle in this in vitro situation. As the authors state, in cells, that happens frequently. This difference is interesting. Why?
    It is customary to refer to polymerized actin as F-actin.
    The supplementary videos are not referenced in the manuscript.

    Significance

    The manuscript describes results from a novel assay to study interactions between F-actin networks and dynamic microtubules in vitro. While of interest to a specialized audience, the overall finding that microtubules can grow into an F-actin meshwork is somewhat incremental especially because of the limited characterization of the F-actin networks used. It remains unclear to what extent this is relevant to a physiological context in cells.

    My field of expertise is related to cytoskeleton dynamics and quantitative microscopy in live cells.

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    Major comments

    In the paper "Microtubules under mechanical pressure can
    breach dense actin networks", the authors showed clear evidence that pressure plays an important role in microtubule breaching into dense actin networks using elegant in vitro reconstitution assays. They have argued that the pressure results from polymerization force of microtubules, which builds up when microtubules are immobilized in the opposite end of breaching, by the means of actin microtubule crosslinking factor Tau.
    It would definitely be interesting to see lack of breaching in the presence of crosslinking deficient Tau construct in order to rule out the off -target effect of Tau on microtubule and actin architecture which may possibly facilitate breaching.

    The authors have also observed microtubule breaching into dense actin networks in living cells. However, in Figure 1C, better cell/ image processing might have been chosen to increase the visibility of actin structures that microtubules encounter on their way to breaching. In Figure S1D, for example, the similar actin structures in lamellipodia are very nicely visible.

    It is also interesting that on Figure 6A, actin bundles look different than the rest of the figures on the paper. It almost looks like actin bundles become branched, whereas in the other Figures actin bundles are either singular or two-three bundles joined together at the point very close to the edge of micropatterned lipid bilayer.

    Minor comments

    In the legend of Figure 4E, it should be written "white arrow" instead of "yellow arrow".
    In the Results section "crosslinking between microtubules and actin bundles increase piercing frequency", in the sentence number 7, it should be written "backwards" instead of "reaward".

    Significance

    The experimental setup of the paper is quite significant in the field, given the difficulty of observing dynamics of dense cytoskeletal structures in living cells. Moreover, the paper gives insight into how microtubule behavior can vary depending on different morphological states of actin network.