Actin network architecture can ensure robust centering or sensitive decentering of the centrosome
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Abstract
The orientation of cell polarity depends on the position of the centrosome, the main microtubule‐organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell‐free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell‐sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture.
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Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Yamamoto and colleagues have investigated the interplay between microtubules (MTs) and actin in positioning the MTOC at "the cell centre". They have developed a novel experimental setup akin to a synthetic cell to study this question. Essentially a cell-sized (15 µm) microwell that is coated in lipid and then tubulin/actin added and the positioning of a MTOC proxy is studied by microscopy. This is a well executed study. These complicated biochemical reconstitutions are the hallmark of Blanchoin and Théry's …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Point-by-point description of the revisions
Black: Comments from reviewers
Green: Answers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Yamamoto and colleagues have investigated the interplay between microtubules (MTs) and actin in positioning the MTOC at "the cell centre". They have developed a novel experimental setup akin to a synthetic cell to study this question. Essentially a cell-sized (15 µm) microwell that is coated in lipid and then tubulin/actin added and the positioning of a MTOC proxy is studied by microscopy. This is a well executed study. These complicated biochemical reconstitutions are the hallmark of Blanchoin and Théry's group, but even so, it's clear that the exact conditions (e.g. tubulin concentration) are fiddly and critical for these experiments to work. The data are clear, well analysed and presented. In brief, the conditions for centring a cytoskeletal network and decentring/polarising it are recapitulated. This is a short, straightforward paper and I found the results to be clear and the authors' interpretation to be well supported by the data.
Two questions occurred to me as I read the paper:
- While the setup is reminiscent of a cell, I suspect that the edge/wall of the microwell is much stiffer than the plasma membrane. So a MT that encounters the wall may behave differently in the cell. This would affect the non-actin conditions but possible also the conditions where an actin mesh is present. Maybe my intuition is not even correct, but I think this issue should be discussed in the paper as a potential limitation of the system.
Author response: We thank the reviewer for this wise comment. Indeed, the deformation of the container may impact the organization of the MT network, the force balance and the final position of the MTOC. We commented this limitation in the revised discussion (page 10 line 31). However, it should be noted that in the presence of a cortical actin network, MTs are much less capable of deforming the cell than in a vesicle or a in cell treated with actin drugs, so our conditions with a cortical actin network are physiologically relevant although the container can not be deformed.
- The graphs in 3C and 4G (lesser extent Fig 1) show nicely that the aMTOC position has apparently rested at a steady state. Some representative trajectories are shown in some figures, but not mentioned much in the text. How does the pathlength (cumulative distance) over time compare to the "distance to centre" measurement? Is there more or less travel under the different conditions? From the supplementary videos it looks like there is a difference. An apparent resting position may still represent significant motion, e.g. circling the centre. What does an analysis of tracklength tell us, if anything?
Author response: We appreciated reviewer’s comment and followed his/her advice. We measured the pathlength (cumulative distance moved) based on the data shown in Figure 3C and 4G. The analysis confirmed that the MTOC was static in the presence of bulk actin network (shown in the new Supplementary Figure 6B). Interestingly, it also showed that the final position adopted by the MTOC in conditions where it could move more freely was also static, as revealed by the saturation of the pathlength after 1 hour. These analyses are shown in the new Supplementary Figure 6B for the centering in the absence of cortical actin, for the non-centering with long microtubules in Supplementary Figure 7E and for the centering with long MTs and a cortical actin network in Supplementary Figure 7E.
Very minor clerical point:
- the first two sentences of the abstract could be clearer. "The position of centrosome, the main microtubule-organizing center (MTOC), is instrumental in the definition of cell polarity. It is defined by the balance of tension and pressure forces in the network of microtubules (MTs)." In the second sentence, "it" and "defined" are confusing. Are you talking about the position of the centrosome or cell polarity?
Author response: We thank the reviewer for this comment. As the reviewer suggested, this was a confusing description. Accordingly, we corrected the sentence in the abstract for :
The orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). It is determined by the balance of tension and pressure forces in the network of microtubules (MTs).
Reviewer #1 (Significance (Required)):
As I see it, the main advance here is in novel experimental setup which has real potential in the field. Existing methods such as MTs inside lipid bubbles are limited, whereas as the microwell method with fabrication methods allows the shape of the "synthetic cell" to be carefully modulated. Tying the results together with cytosim simulations is also a powerful combination. There is a lot of interest in bottom-up reconstitution of cell biological phenomena, especially those that underlie specialised cell processes, e.g. polarity. My expertise: microtubules in a cellular context with limited experience of MT reconstitution assays.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: This manuscript describes the use of an elegant in vitro reconstitution system to study the effect of variations in the organization of the actin network on the positioning of a microtubule organizing center (MTOC) within the cell. By using a reconstituted system the authors are able to specifically study the contribution of the "pushing" forces generated by microtubule (MT) growth, without the confounding influence of other factors, like pulling forces from MT motors. The authors find that a bulk actin networks at sufficient density can impair MTOC displacement, likely a result of the large viscous drag of the MTOC. Next they show that MTOC centering more resilient to changes in microtubule length. Finally they show that an asymmetric actin network can cause asymmetric positioning of the MTOC.
Major comments:
- The model the authors put forth is that the growth of long MTs leads to decentering as a result of the MTs slipping along the well edge. The presence of a cortical actin mesh prevents this slipping. Their argument would be strengthened with and analysis of the MT behaviors in the various conditions. For example when discussing MTOC in well without actin...
"As they grew, they first ensured a proper centering but after an hour, MT elongation and slippage along microwell edges broke the network symmetry and MTs pushed aMTOC away from the center (Figure 1I, J and Supplementary Movie 2)"
In this movie I don't see evidence of MTs hitting the cortex and sliding on the "short" side of the well relative to the MTOC. An analysis of the behavior of MTs in various circumstances would help link the behavior of MTs to the movement of the MTOC for all of their conditions. What fraction of MTs hit the cortex and remain relatively motionless, what fraction slide, what fraction catastrophe, what fraction turn and follow the curve of the well? And how does this behavior change for microtubules that end up on the short side vs. the long side of the MTOC? This type of analysis would solidify their model for how centering/decentering occurs in the various conditions they test.
Author response: This is a fair criticism. The possibility to perform fine analysis of MT dynamics is technically limited by the fluorescent background due to free tubulin dimers. It is the reason why classical in vitro assays are monitored in TIRF microscopy, which is not possible here since MTOCs move in 3D in the microwells. In addition, working with higher laser power to increase the signal to noise ratio generates severe photodamages on MTs. Nevertheless, we could visualize MT dynamics and displacements near the edge of the microwells and describe their behavior more precisely than in the previous version of our manuscript. New images and tracking of MT behavior are now reported in the new Figure 4E, 4F and 5G, as well as the new supplementary Figure 4C, 4D, 7B, and 7C. We also replaced the supplementary movie 2 and Figure 1I in order to show more clearly MTs hitting and slipping along the well boundary. In addition, we also characterized the pivoting of MTs around the MTOC and near the edge of the microwell in order to better characterize the effect of cortical actin. This is now shown in the new Figure 4G and 4H as well as in the new Supplementary Figure 7C-D). We found that the changes in MT orientation and position, at the centrosome and at the contact with the microwell, were clearly prevented by the presence of cortical actin.
- The authors use simulations to support their in vitro findings. However, their simulations have many more microtubules emanating from the MTOC than their experiment (Looks like about 50 in the cytosim and they state they are aiming for 15-20 in the aMTOCs). Do the simulations still reproduce the behavior of the in vitro system with a similar number of MTs?
Author response: This is another fair criticism. We addressed this point by performing simulations with 10~30 microtubules (the number of MTs is variable because of MT dynamics) which are more similar to the number of MTs that we obtained in our experimental conditions. Results were consistent with previous simulations with higher number of MTs and are now shown in the new supplementary figures 6E-F, 7G and 8I).
- When the actin networks are asymmetric, the authors see decentering of the MTOC towards the side with less actin. However there is still actin on the side where the MTOC will move to and in some of their images it looks pretty think. Is the actin on that side not dense enough to prevent MT sliding along the "cortex"? If so, can they generate less dense, but uniform actin networks on the "cortex", where MTs can slide. Again descriptions of MT behaviors would be useful in understanding what is happening.
Author response: We thank the reviewer for asking this important question. We followed reviewer’s advice and generated homogeneous and less dense cortex by working at lower concentration of actin (0.5 mM). In such conditions, we could not see the centering effect that was observed with dense cortex. These new data are now shown in the new Supplementary Figure 7I. This effect was also tested with numerical simulations (new Supplementary Figure 7J) which were consistent with the key role played by actin network density for MT network positioning by cortical friction.
Minor Comments: 1)Title - the current title implies that actin is balancing the forces generated by the MTs. I'm not sure this is a good description of what is shown in the paper.
Author response: We thank the reviewer for pointing at this issue. We revised the title to:
Reconstitution of centrosome positioning by the production of pushing forces in microtubules growing against the actin network.
2)The discussion would benefit from more explanation about how the results of this paper relate to the classic examples of MTOC positioning they cite. How do they envision the actin and MTs interacting in these systems and what new insight have we gained from the experiments in this manuscript.
Author response: This is a good suggestion. We added some comments in our discussion about the actin network asymmetry in several classical examples of cell polarization and explained how our observations suggest some new interpretation on the role of this asymmetry in the reorganization of forces in the MT network and on the consequential peripheral positioning of the MTOC.
Reviewer #2 (Significance (Required)):
Overall, this work is a significant advance in our understanding of the potential mechanisms of MTOC movement in cells via pushing by MT growth. The experimental system they have developed is powerful advance, allowing meaningful MTOC reconstitution experiments to be performed in chambers of approximately cellular size. This is an important contribution to understanding the interaction between microtubule pushing and the actin cortex.
Reviewer expertise: Cell biology of MTOC assembly and positioning. I do not have the expertise to assess the parameters used to generate their cytosim models.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Review of "The architecture of the actin network can balance the pushing forces produced by growing microtubules" by Yamamoto et al.
The means by which cells maintain their characteristic cytoskeletal architectures is not well understood. This is in part because there is considerable variation in such architectures with, for example, fibroblasts, neurons, and epithelial cells. It is also in part because the microtubule, actin and intermediate filaments engage in a wide range of mechanical and signaling crosstalk mediated by a wealth of proteins and signaling networks, which further complicates the picture.
In the current study, Yamamoto take the welcome step of developing a simplified system for assessing the mutual contributions of microtubules and F-actin for general cytoskeletal organization in vitro (specifically, in lipid-lined microwells). This allows them to define basic principles of microtubule-F-actin interactions in the absence of the various confounding factors alluded to above. Using their model, they show that artificial MTOCs (aMTOCs) alone will center but as a complex function of microtubule length (controlled by varying tubulin concentrations). That is, the aMTOCs are randomly positioned with short microtubules, stably centered with intermediate length microtubules, and randomly oriented with very long microtubules (following symmetry breaking).
They then assess the contributions of F-actin to the centering process. In low concentrations of "bulk" F-actin (ie F-actin distributed throughout the droplet) there is no effect on centering whereas at higher concentrations of bulk F-actin, centering is impaired as is the translocation of the aMTOCs. In the presence of uniform peripheral F-actin, in contrast, aMTOC centering is enhanced, and rendered less sensitive to variations in microtubule length. Finally, when the authors contrive a situation in which the peripheral F-actin is non-uniform (by lowering the concentration of actin and adding alpha-actinin, which creates a peripheral ring of F-actin with (I think) relatively less F-actin within the ring), the aMTOCs position themselves within the ring.
Finally, the authors extend their results with simulations that indicate that the various behaviors can be explained by a combination of friction, pushing and slippage.
This study is fascinating and will be of general interest to anyone who seeks to understand the contributions of mechanical forces to cytoskeletal organization in a minimal system. I have only minor concerns; these are listed below.
- Some of the terminology was a little confusing. The authors introduce the term "inner zone" (pg. 8) without defining it. From the context, it seems like they are talking about the approximate center of the ring of peripheral F-actin. If so, why not just do away with the term "inner zone" and refer to the ring center. If it isn't the ring center, then more explanation is needed as to what the inner zone actually is.
Author response: We apologize for this confusion and appreciate reviewer’s comment. We coined earlier the term “actin inner zone” to define the central cytoplasmic region in cells that is devoid of actin filament (Jimenez et al., Current Biology, 2021). Because it was a confusing point, we clarified this in the revised version of the manuscript (Page 8, Line 20). What we would like to call the “inner zone” is the region inside of the actin cortex. The definition of this zone and of its geometrical reference points were also pictured more precisely in the new Supplementary Figure 9B.
- It is not clear from the text or the images if the region within the F-actin ring has less F-actin, more F-actin, or the same amount of F-actin as the region outside the F-actin ring. This point should be clarified, as it makes a big difference in the interpretation of the findings.
Author response: We apologize for this lack of clarity. In the revised version of our manuscript, we plotted a line scan intensity profile of the actin fluorescence (new Supplementary Figure 9B). It showed that the region within the actin inner zone contained much less actin than in the cortex. This is consistent with our interpretation of a region-selective pattern of friction acting on microtubules.
- Ideally, the authors would include manipulations in which the high concentration of peripheral F-actin is combined with alpha-actinin because, as currently presented, the authors are drawing conclusions from changing two variables at once (ie going from a high concentration of peripheral F-actin to a lower concentration with added alpha-actinin). Thus, the authors cannot cleanly distinguish between effects that arise from F-actin asymmetry versus the presence of an F-actin crosslinker. Since the crosslinking is likely to change the mechanical properties of the peripheral F-actin network, this point should at least be addressed in the text, if not by experiments.
Author response: We are not sure to fully understand the reviewer’s point. We don’t understand how the crosslinking of a symmetric actin network could break the symmetry of the MT network and force its off-centering. The opposite is clearer to us. A homogeneous and loose actin network can allow MT gliding and MTOC off-centering (like in in Supplementary Figure 7J). The mechanical reinforcement of this network by crosslinkers could indeed resist gliding. But the consequence of this resistance would be similar to the consequence of a dense network: a more robust centering (like in Figure 4). So we don’t understand how the crosslinking by alpha-actinin, rather than the asymmetry of the actin network, could be at the origin of the off-centering we observed. In addition the off-centering of the MTOC was systematically aligned with the asymmetry of the actin network, so both parameters were clearly connected.
Reviewer #3 (Significance (Required)):
This is an elegant, well-designed study that provides a clear description of how basic mechanical forces can contribute to cytoskeletal organization in a simplified model system.
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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
Review of "The architecture of the actin network can balance the pushing forces produced by growing microtubules" by Yamamoto et al.
The means by which cells maintain their characteristic cytoskeletal architectures is not well understood. This is in part because there is considerable variation in such architectures with, for example, fibroblasts, neurons, and epithelial cells. It is also in part because the microtubule, actin and intermediate filaments engage in a wide range of mechanical and signaling crosstalk mediated by a wealth of proteins and signaling networks, which further complicates the picture.
In the current study, …
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 #3
Evidence, reproducibility and clarity
Review of "The architecture of the actin network can balance the pushing forces produced by growing microtubules" by Yamamoto et al.
The means by which cells maintain their characteristic cytoskeletal architectures is not well understood. This is in part because there is considerable variation in such architectures with, for example, fibroblasts, neurons, and epithelial cells. It is also in part because the microtubule, actin and intermediate filaments engage in a wide range of mechanical and signaling crosstalk mediated by a wealth of proteins and signaling networks, which further complicates the picture.
In the current study, Yamamoto take the welcome step of developing a simplified system for assessing the mutual contributions of microtubules and F-actin for general cytoskeletal organization in vitro (specifically, in lipid-lined microwells). This allows them to define basic principles of microtubule-F-actin interactions in the absence of the various confounding factors alluded to above. Using their model, they show that artificial MTOCs (aMTOCs) alone will center but as a complex function of microtubule length (controlled by varying tubulin concentrations). That is, the aMTOCs are randomly positioned with short microtubules, stably centered with intermediate length microtubules, and randomly oriented with very long microtubules (following symmetry breaking).
They then assess the contributions of F-actin to the centering process. In low concentrations of "bulk" F-actin (ie F-actin distributed throughout the droplet) there is no effect on centering whereas at higher concentrations of bulk F-actin, centering is impaired as is the translocation of the aMTOCs. In the presence of uniform peripheral F-actin, in contrast, aMTOC centering is enhanced, and rendered less sensitive to variations in microtubule length. Finally, when the authors contrive a situation in which the peripheral F-actin is non-uniform (by lowering the concentration of actin and adding alpha-actinin, which creates a peripheral ring of F-actin with (I think) relatively less F-actin within the ring), the aMTOCs position themselves within the ring.
Finally, the authors extend their results with simulations that indicate that the various behaviors can be explained by a combination of friction, pushing and slippage.
This study is fascinating and will be of general interest to anyone who seeks to understand the contributions of mechanical forces to cytoskeletal organization in a minimal system. I have only minor concerns; these are listed below.
Some of the terminology was a little confusing. The authors introduce the term "inner zone" (pg. 8) without defining it. From the context, it seems like they are talking about the approximate center of the ring of peripheral F-actin. If so, why not just do away with the term "inner zone" and refer to the ring center. If it isn't the ring center, then more explanation is needed as to what the inner zone actually is.
It is not clear from the text or the images if the region within the F-actin ring has less F-actin, more F-actin, or the same amount of F-actin as the region outside the F-actin ring. This point should be clarified, as it makes a big difference in the interpretation of the findings.
Ideally, the authors would include manipulations in which the high concentration of peripheral F-actin is combined with alpha-actinin because, as currently presented, the authors are drawing conclusions from changing two variables at once (ie going from a high concentration of peripheral F-actin to a lower concentration with added alpha-actinin). Thus, the authors cannot cleanly distinguish between effects that arise from F-actin asymmetry versus the presence of an F-actin crosslinker. Since the crosslinking is likely to change the mechanical properties of the peripheral F-actin network, this point should at least be addressed in the text, if not by experiments.
Significance
This is an elegant, well-designed study that provides a clear description of how basic mechanical forces can contribute to cytoskeletal organization in a simplified model system.
-
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
Summary:
This manuscript describes the use of an elegant in vitro reconstitution system to study the effect of variations in the organization of the actin network on the positioning of a microtubule organizing center (MTOC) within the cell. By using a reconstituted system the authors are able to specifically study the contribution of the "pushing" forces generated by microtubule (MT) growth, without the confounding influence of other factors, like pulling forces from MT motors. The authors find that a bulk actin networks at sufficient density can impair MTOC displacement, likely a result of the large viscous drag of the MTOC. Next …
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
Summary:
This manuscript describes the use of an elegant in vitro reconstitution system to study the effect of variations in the organization of the actin network on the positioning of a microtubule organizing center (MTOC) within the cell. By using a reconstituted system the authors are able to specifically study the contribution of the "pushing" forces generated by microtubule (MT) growth, without the confounding influence of other factors, like pulling forces from MT motors. The authors find that a bulk actin networks at sufficient density can impair MTOC displacement, likely a result of the large viscous drag of the MTOC. Next they show that MTOC centering more resilient to changes in microtubule length. Finally they show that an asymmetric actin network can cause asymmetric positioning of the MTOC.
Major comments:
- The model the authors put forth is that the growth of long MTs leads to decentering as a result of the MTs slipping along the well edge. The presence of a cortical actin mesh prevents this slipping. Their argument would be strengthened with and analysis of the MT behaviors in the various conditions. For example when discussing MTOC in well without actin...
"As they grew, they first ensured a proper centering but after an hour, MT elongation and slippage along microwell edges broke the network symmetry and MTs pushed aMTOC away from the center (Figure 1I, J and Supplementary Movie 2)"
In this movie I don't see evidence of MTs hitting the cortex and sliding on the "short" side of the well relative to the MTOC. An analysis of the behavior of MTs in various circumstances would help link the behavior of MTs to the movement of the MTOC for all of their conditions. What fraction of MTs hit the cortex and remain relatively motionless, what fraction slide, what fraction catastrophe, what fraction turn and follow the curve of the well? And how does this behavior change for microtubules that end up on the short side vs. the long side of the MTOC? This type of analysis would solidify their model for how centering/decentering occurs in the various conditions they test.
The authors use simulations to support their in vitro findings. However, their simulations have many more microtubules emanating from the MTOC than their experiment (Looks like about 50 in the cytosim and they state they are aiming for 15-20 in the aMTOCs). Do the simulations still reproduce the behavior of the in vitro system with a similar number of MTs?
When the actin networks are asymmetric, the authors see decentering of the MTOC towards the side with less actin. However there is still actin on the side where the MTOC will move to and in some of their images it looks pretty think. Is the actin on that side not dense enough to prevent MT sliding along the "cortex"? If so, can they generate less dense, but uniform actin networks on the "cortex", where MTs can slide. Again descriptions of MT behaviors would be useful in understanding what is happening.
Minor Comments:
Title - the current title implies that actin is balancing the forces generated by the MTs. I'm not sure this is a good description of what is shown in the paper.
The discussion would benefit from more explanation about how the results of this paper relate to the classic examples of MTOC positioning they cite. How do they envision the actin and MTs interacting in these systems and what new insight have we gained from the experiments in this manuscript.
Significance
Overall, this work is a significant advance in our understanding of the potential mechanisms of MTOC movement in cells via pushing by MT growth. The experimental system they have developed is powerful advance, allowing meaningful MTOC reconstitution experiments to be performed in chambers of approximately cellular size. This is an important contribution to understanding the interaction between microtubule pushing and the actin cortex.
Reviewer expertise: Cell biology of MTOC assembly and positioning. I do not have the expertise to assess the parameters used to generate their cytosim models.
-
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
Yamamoto and colleagues have investigated the interplay between microtubules (MTs) and actin in positioning the MTOC at "the cell centre". They have developed a novel experimental setup akin to a synthetic cell to study this question. Essentially a cell-sized (15 µm) microwell that is coated in lipid and then tubulin/actin added and the positioning of a MTOC proxy is studied by microscopy. This is a well executed study. These complicated biochemical reconstitutions are the hallmark of Blanchoin and Théry's group, but even so, it's clear that the exact conditions (e.g. tubulin concentration) are fiddly and critical for these …
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
Yamamoto and colleagues have investigated the interplay between microtubules (MTs) and actin in positioning the MTOC at "the cell centre". They have developed a novel experimental setup akin to a synthetic cell to study this question. Essentially a cell-sized (15 µm) microwell that is coated in lipid and then tubulin/actin added and the positioning of a MTOC proxy is studied by microscopy. This is a well executed study. These complicated biochemical reconstitutions are the hallmark of Blanchoin and Théry's group, but even so, it's clear that the exact conditions (e.g. tubulin concentration) are fiddly and critical for these experiments to work. The data are clear, well analysed and presented. In brief, the conditions for centring a cytoskeletal network and decentring/polarising it are recapitulated. This is a short, straightforward paper and I found the results to be clear and the authors' interpretation to be well supported by the data.
Two questions occurred to me as I read the paper:
- While the setup is reminiscent of a cell, I suspect that the edge/wall of the microwell is much stiffer than the plasma membrane. So a MT that encounters the wall may behave differently in the cell. This would affect the non-actin conditions but possible also the conditions where an actin mesh is present. Maybe my intuition is not even correct, but I think this issue should be discussed in the paper as a potential limitation of the system.
- The graphs in 3C and 4G (lesser extent Fig 1) show nicely that the aMTOC position has apparently rested at a steady state. Some representative trajectories are shown in some figures, but not mentioned much in the text. How does the pathlength (cumulative distance) over time compare to the "distance to centre" measurement? Is there more or less travel under the different conditions? From the supplementary videos it looks like there is a difference. An apparent resting position may still represent significant motion, e.g. circling the centre. What does an analysis of tracklength tell us, if anything?
Very minor clerical point:
- the first two sentences of the abstract could be clearer. "The position of centrosome, the main microtubule-organizing center (MTOC), is instrumental in the definition of cell polarity. It is defined by the balance of tension and pressure forces in the network of microtubules (MTs)." In the second sentence, "it" and "defined" are confusing. Are you talking about the position of the centrosome or cell polarity?
Significance
As I see it, the main advance here is in novel experimental setup which has real potential in the field. Existing methods such as MTs inside lipid bubbles are limited, whereas as the microwell method with fabrication methods allows the shape of the "synthetic cell" to be carefully modulated. Tying the results together with cytosim simulations is also a powerful combination. There is a lot of interest in bottom-up reconstitution of cell biological phenomena, especially those that underlie specialised cell processes, e.g. polarity.
My expertise: microtubules in a cellular context with limited experience of MT reconstitution assays.
-