A B-cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation

Article activity feed

1. 

   Version published to 10.7554/elife.72805 on eLife

   Apr 11, 2022
2. 

   Version published to 10.1101/2021.08.12.456133v2 on bioRxiv

   Feb 10, 2022
3. 

   eLife

   Oct 5, 2021

   Author Response:

   Reviewer #1:

   A role for integrins in lowering the threshold for B cell activation was first observed over 15 years ago, but the mechanism has remained elusive. In this paper, Wang et al. investigate the role of LFA-1:ICAM-1 ligation in B cell synapse formation using live-cell super-resolution fluorescence microscopy in both primary B cells and the A20 B cell line. The use of super-resolution imaging is critical to the investigation as it reveals a level of organisation of the actomyosin network that is not visible with conventional microscopy approaches such as TIRF microscopy. They find that LFA-1:ICAM-1 ligation promotes the formation of actomyosin arcs that regulate various activities in the B cell synapse including BCR signalling, BCR:antigen microcluster transport, and the centralisation of antigen. In agreement …

   Author Response:

   Reviewer #1:

   A role for integrins in lowering the threshold for B cell activation was first observed over 15 years ago, but the mechanism has remained elusive. In this paper, Wang et al. investigate the role of LFA-1:ICAM-1 ligation in B cell synapse formation using live-cell super-resolution fluorescence microscopy in both primary B cells and the A20 B cell line. The use of super-resolution imaging is critical to the investigation as it reveals a level of organisation of the actomyosin network that is not visible with conventional microscopy approaches such as TIRF microscopy. They find that LFA-1:ICAM-1 ligation promotes the formation of actomyosin arcs that regulate various activities in the B cell synapse including BCR signalling, BCR:antigen microcluster transport, and the centralisation of antigen. In agreement with earlier studies, they show that LFA-1:ICAM-1 ligation is required for B cells to centralise antigen that is present at very low density. They also demonstrate that myosin IIa contractility is required for the formation of the actomyosin arcs and promotes the exertion of strong traction forces on the antigen- and ICAM-1-presenting substrate. Using a small molecule inhibitor of formin activity in combination with miRNA knockdown of the formin mDia1, the authors show that the actomyosin arcs originate at the outer edge of the synapse and that their generation is formin dependent. These data provide a much-needed advance to our understanding of the role LFA-1 plays in the earliest events in B cell responses to antigen.

   The conclusions of the paper are mostly well supported by the data, but there are a few points that would need to be clarified.

   1. The requirement for LFA-1:ICAM-1 ligation in the formation of the actomyosin arcs is not clear. The authors observe that ~30% of B cells form actomyosin arcs with anti-IgM stimulation only (Figure 1). Does LFA-1:ICAM-1 ligation simply stabilise the arcs and therefore make their appearance more likely, or does it promote the formation of a distinct actomyosin network with unique functions? The images and videos selected to represent cells stimulated with anti-IgM only (Figure 1; Movies 1A and 1B) seem form a highly branched actin network throughout the synapse, but it would be informative to see cells having the actomyosin arcs for comparison. Since B cells stimulated with anti-IgM alone are capable of signalling and centralising antigen, it would be interesting to know whether and how these two populations (with and without arcs) differ.

   We thank the reviewers for their questions regarding this central aspect of our study. In response to the reviewers’ statement “The requirement for LFA-1:ICAM-1 ligation in the formation of the actomyosin arcs is not clear”, our results state that “Consistently, scoring B cells for the presence of a discernable actin arc network showed that the addition of ICAM-1 increases the percentage of such cells from ~30% to ~70% (Fig. 1G).” Importantly, we then state that “dynamic imaging showed that the arcs in cells engaged with anti-IgM alone are typically sparse and transient (Movies 1A and 1B), while those in cells engaged with both anti-IgM and ICAM-1 are dense and persistent (Movies 2A and 2B).” To emphasize this point, which we think is clear when comparing Movies 1A/1B to Movies 2A/2B, we have now added the following two sentences to the text: “In other words, when B cells receiving only anti-IgM stimulation do form discernable arcs (see, for example, those marked by magenta arrows in Fig. 1A and 1B), they are much sparser and less robust than those formed by cells also receiving ICAM-1 stimulation. Moreover, we never saw even one B cell receiving anti-IgM stimulation alone that possessed a robust actin arc network.” Please note that the magenta arrows in Fig. 1A and 1B were added upon revision. In summary, the cell shown in Fig. 1E, which lacks discernable arcs, is representative of ~70% of anti-IgM stimulated cells, while the cell shown in Fig. 1F, which possesses a robust arc network, is representative of ~70% of anti-IgM+ICAM-1 stimulated cells.

   We would also like to address what we think is a misunderstanding regarding our images in Figure 1, as reflected in reviewer 1’s statement: “The images and videos selected to represent cells stimulated with anti-IgM only (Figure 1; Movies 1A and 1B) seem form a highly branched actin network throughout the synapse”. The outer, Arp2/3-generated, branched network comprising the dSMAC/lamellipodium in primary B cells is really quite thin under both stimulation conditions (please see Fig. 1, E1, E2, F1 and F2). In other words, we would not characterize the region between this thin, outer, canonical branched actin network and the central actin hypodense area (i.e. the region corresponding to the pSMAC) in B cells engaged with anti-IgM alone as “a highly branched actin network throughout”. We described it in the text as “a highly disorganized mixture of short actin filaments/fibers and actin foci”. While it likely contains some branched filaments, it is not a canonical branched actin network like the one comprising the dSMAC. Indeed, it is a lot like the mixture of actin asters, actin foci, branched actin and linear filaments described in Hela cells using the same imaging technique ((Fritzsche et al., 2017); we have now cited this paper). Of note, A20 B cells make a much bigger branched actin/dSMAC/lamellipodium than do primary B cells (compare the image of the representative A20 B cell in Fig. 1J to the various images of primary B cells in this figure). Interestingly, this difference between immortalized cells and primary cells is conserved in T cells, as Jurkat T cells make a much bigger branched actin/dSMAC/lamellipodium than do primary T cells (Murugesan et al, JCB 2016).

   Although the reviewers did not specifically comment on why only ~70% of primary B cells engaged with both anti-IgM and ICAM-1 make actomyosin arcs, we note that this is also the case for both Jurkat T cells and primary T cells (Murugesan et al, JCB 2016). We do not know why the number does not go to 100%, but the ~70% limit is the case for both B cells and T cells. Of note, in unpublished work we see that LFA-1 ligation also promotes actomyosin arc formation in T cells.

   With regard to the reviewers’ question “Does LFA-1:ICAM-1 ligation simply stabilize the arcs and therefore make their appearance more likely, or does it promote the formation of a distinct actomyosin network with unique functions?”, we think that ICAM-1 engagement likely leads to the strong activation of RhoA, which then serves to drive both the formation of actin arcs by recruiting, unfolding, and activating mDia at the plasma membrane, and the stabilization and concentric organization of these arcs by activating myosin 2A filament assembly and contractility. In other words, we think ICAM-1 engagement leads simultaneously to the creation and stabilization/organization of the arcs. While it is true that BCR stimulation alone activates RhoA signaling to some extent (see Saci and Carpenter, Mol Cell 2005 and Caloca et al, J Biol Chem 2008), and that this may account for the sparse actin arcs seen in cells stimulated with anti-IgM alone, it is likely that RhoA signaling is more robust with the addition of integrin co-stimulation (Lawson & Burridge, 2014) and that this would promote the creation of the actomyosin arcs seen in these cells. That said, without independent measures of the creation and stabilization/turnover of the arcs, we cannot gauge the relative significance of creation versus stabilization/turnover in determining the steady state amount of arcs. To address this limitation, we have added the following sentence to the section of the Discussion dealing with integrin-dependent signaling pathways leading to actomyosin arc formation: “Finally, future studies should also seek to clarify the extent to which integrin ligation promotes the formation of actomyosin arcs by driving their creation versus stabilizing them once created.

   With regard to the reviewers’ comment that “B cells stimulated with anti-IgM alone are capable of signalling and centralising antigen” we would like to emphasize that our study focuses on B cell immune synapse formation under limiting antigen conditions, where a previous study (Carrasco et al. Immunity 2004) and our data in Fig. S5 show that the impairments in BCR signaling and antigen centralization seen under this condition are rescued by integrin co-stimulation. We expand upon these findings by showing in Figures 5 and 6 that this integrin-dependent rescue of antigen centralization and BCR signaling requires actomyosin. In other words, the actomyosin arc network described here is required for integrin co-stimulation to promote antigen centralization and signaling under limiting antigen conditions. We agree with the reviewer that under non-limiting antigen conditions B cells can signal and centralize antigen in the absence of ICAM-1. That said, these high levels of BCR stimulation are probably not as physiological as limiting BCR stimulation. Finally, our data in Figure S7 shows that antigen centralization in primary B cells receiving non-limiting anti-IgM stimulation alone is also significantly impaired when myosin is inhibited. This suggests that cells receiving high levels of BCR stimulation employ myosin in some fashion to drive antigen centralization. We now close the section describing these results with the following statement: “That said, additional experiments should help define exactly how myosin contributes to antigen centralization in B cells receiving only strong anti-IgM stimulation."

   Finally, and most generally, we avoided the use of the word “requirement” as in the reviewer’s statement “the requirement for LFA-1:ICAM-1 ligation in the formation of the actomyosin arcs is not clear”. Given that some B cells receiving only anti-IgM stimulation create arcs (albeit sparse and transient), we were careful to say throughout the text that ICAM-1 engagement “promotes” actomyosin arc formation. We think our evidence for this is compelling.

   1. The authors propose that the contractile actomyosin network formed in the presence of LFA-1:ICAM-1 interactions promotes B cell activation especially at low antigen concentrations; however, their data focus only on early signalling (pCD79a and pCD19) and it would be helpful to know whether LFA-1:ICAM-1 interactions impact signalling further downstream.

   We thank the reviewer for this important suggestion, which we will address in a future study.

   1. The observation that some GC B cells centralise antigen is very interesting, but there are a few aspects of this investigation that should be expanded upon. The authors show that with LFA-1:ICAM-1 interactions, GC B cells are about equally likely to organise BCR:antigen complexes into peripheral clusters and centralised clusters. It would be informative to have, in the same study (Figure 7), a comparison with GC B cells stimulated with antigen alone. The reason is that other studies investigating GC B cell synapse architecture did not quantify antigen organisation in this way, so it is difficult to make comparisons with previous work. It would also be very useful to see how the actomyosin network is organised in GC B cells exhibiting different synaptic architectures (i.e. peripheral versus central clusters), especially given the critical role of myosin IIa activity in GC B cell antigen affinity discrimination. Additionally, while it is a very interesting observation that LFA-1:ICAM-1 interactions may affect GC B cell synapse organisation, it is not clear whether this has an impact on cellular function. For instance, does antigen and actomyosin organisation in GC B cell synapses contribute to differences in signalling or traction force generation? In the introduction the authors suggest that actomyosin arcs contribute to antibody affinity maturation (line 87-88), but without functional studies to support this claim I think it is too speculative.

   We thank the reviewer for their comments and suggestions regarding our GC data. Our sole purpose in performing the experiments in Figure 7 was to see if GC B cells can also make actomyosin arcs. We did this because recent papers and reviews state that the organization and dynamics of actin at GC B cell synapses are completely different from the organization and dynamics of actin at naive B cells synapses. As such, these initial observations are meant to add to previous work on GC B cells rather than generate direct comparisons. The reviewers appear to agree that the data in Figure 7 shows convincingly that a subset of GC B cells can make actomyosin arcs that are indistinguishable in appearance from those formed by naive B cells (so the specific claim we are making does not “require additional supporting data”). Rather, the reviewers request that we expand on the data in Figure 7 in several ways, some of which we had already mentioned in the Discussion (“While additional work is required to prove that the subset of GC B cells with actomyosin arcs are the ones that centralize antigen, this seems likely given our evidence here that actomyosin arcs drive antigen centralization in naïve B cells.”, and “Future work will also be required to understand why GC B cells vary with regard to actomyosin organization and the ability to centralize antigen 18 (e.g. dark zone versus light zone GCs)”). In addition to these statements, we now end the section describing the results in Figure 7 with the following statement: “We note, however, that our conclusions regarding actomyosin arcs in GC B cells require additional supporting data that include testing the ICAM-1 dependence of actomyosin arc formation and quantitating the contributions that this contractile structure makes to GC B cell traction force, signaling, and antigen centralization.”

   With regard to the reviewers concerns indicated by their comment “In the introduction the authors suggest that actomyosin arcs contribute to antibody affinity maturation (line 87-88), but without functional studies to support this claim I think it is too speculative”, we have changed the relevant sentence to “Finally, we show that germinal center (GC) B cells can also create this actomyosin structure, suggesting that it may contribute to the functions of GC B cells as well”.

   Reviewer #2:

   The manuscript utilizes elegant imaging tools to describe the contractile actomyosin arcs, induced by integrin-ligation, and their involvement in antigen gathering in B cells. The findings are important and have the potential to make a considerable impact in the field. The main conclusions are well supported by strong data and the manuscript convincingly brings across the need of integrin-ligation to induce generation of the arc network and the role of this structure in antigen gathering. The methods and the quality of imaging are state-of-the-art and provide an important example for future studies in B cell immune synapse. Some aspects of the study would benefit from clarification and extended experimentation or analysis.

   1. In addition to cultured B cells, the work includes naïve primary B cells as well as isolated germinal center B cells. While the use of primary cells adds value to the study, in most cases the cells are activated first with LPS prior to transfection with F-Tractin constructs. Such a treatment is likely to alter the cytoskeletal features of the naïve B cells and, thus, it would be informative to provide an analysis of this effect.

   We thank the reviewer for commenting on this. To clarify, we treated primary B cells with LPS to promote cell survival during the harsh nucleofection/electroporation conditions that otherwise kill these fragile cells. Moreover, the cells were rested for 24 hours post-nucleofection in the absence of LPS to promote return to a resting state, as previously described (see(Freeman et al., 2011)). Moreover, only those primary B cells used for live cell imaging of the F-actin using the F-actin reporter F-Tractin were LPS treated. The majority of our experiments employed non-treated ex vivo B cells that were fixed, stained and imaged for quantitation. Importantly, under conditions of ICAM-1 co-stimulation, the actomyosin arcs formed by ex vivo B cells and by LPS-activated cells were indistinguishable. For example, compare the F-Tractin-expressing cell in Fig. 3A to the non-treated cells in Fig. 3D and Fig. 7A. To summarize, then, only live-cell imaging experiments that required F-Tractin to visualize F-actin dynamics were performed using LPS-activated B cells. Finally, we clarified in the Methods that we refer to all primary B cells as “naïve” B cells because they had not been previously activated by antigen at the time of antigen stimulation.

   Reviewer #3:

   The work 'A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation' by Wang et al. describes the importance of integrin mediated B-cell co-stimulation for IS formation in B-cells by fostering the formation of myosin II A driven actin arcs that are essential in the transport of IgM clusters towards the IS center.

   The work presented here, i.e. experiments and analysis, is very thoroughly done and includes tests and controls using different labelling strategies and constructs of myosin II A, multiple cell types including primary cells and a range of chemical inhibitors to rule out artefacts.

   The authors claim that the observation of actin arcs in B-cells co-stimulated by ICAM-1 - LFA-1 interaction is important for the efficient activation of B-cells in the presence of limiting levels of anti-IgM and this is very well supported by the experiments. However, it was a bit surprising that the paper did not draw much of parallels between the observed phenomenon and the reported actin arcs in activated T-cells even though some of the authors were very much involved in such work on T-cells. If there is a good reason to believe there is no ground to draw comparisons, this would then also need to be highlighted by the authors.

   We thank the reviewer for their comments. We have now added the following two sentences to the Discussion: “It is also important to note that the contractile actomyosin arcs described here in B cells and the actomyosin arcs described previously in T cells (Murugesan et al., 2016) share much in common as regards formation, organization and dynamics (Hammer et al., 2019; Wang & Hammer, 2020). Going forward, it will be vital to define how these two immune cell types harness the same contractile synaptic structure to accomplish different goals (i.e. antibody production by B cells and target cell killing by T cells).”

   The work on establishing the drivers of actin arc formation and dynamics is well done, but it is important to note that previous work has analyzed actin arc formation in other cell types. Work by Bershadsky has already established many 'ground rules' for the formation of actin arcs and the role of integrin adhesion, formin activity and myosin II in the process (Tee YH, Shemesh T, Thiagarajan V, Hariadi RF, Anderson KL, Page C, Volkmann N, Hanein D, Sivaramakrishnan S, Kozlov MM, Bershadsky AD. 2015. Cellular chirality arising from the self-organization of the actin cytoskeleton. Nat Cell Biol 17:445-457. doi:10.1038/ncb3137). It might be very instructive if the authors could put their findings in relation to this work.

   The formation of actin arcs is also well studied in U2OS cells and the results presented here could highlight interesting general features of this process observed in very different cell types (Tojkander S, Gateva G, Husain A, Krishnan R, Lappalainen P. 2015. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly. Elife 4:1-28. doi:10.7554/eLife.06126; Bur-nette DT, Shao L, Ott C, Pasapera AM, Fischer RS, Baird MA, Der Loughian C, Delanoe-Ayari H, Paszek MJ, Davidson MW, Betzig E, Lippincott-Schwartz J. 2014. A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells. J Cell Biol 205:83-96. doi:10.1083/jcb.201311104).

   In this regard, the findings about the importance of myosin II A activity, integrin adhesion and mDia1 in the formation of actin arcs is not that surprising and the authors might rather highlight the important role of these newly studied structures for co-stimulation in B-cells as this is the more novel and insightful bit of the work.

   We thank the reviewer for their comments. Indeed, our prior work in T cells (Murugesan et al., 2016; Yi et al., 2012) also linked formin activity and myosin 2 contractility to the formation of actin arcs and the generation of integrin-based adhesion. We now cite the papers highlighted by the reviewer using the following sentence in the revised Discussion: “It is important to note here that several earlier studies performed using other cell types have also linked formin activity and myosin 2 contractility to the formation of actin arcs and the generation of integrin-based adhesions (Burnette et al., 2014; Tee et al., 2015; Tojkander et al., 2015).” As for highlighting the relevance of our results for the B cell field, we think we have done that by demonstrating the existence of this contractile network in B cells, and by showing that it provides mechanistic insight into how integrin co-stimulation promotes synapse formation and B cell activation when antigen is limiting. Given that many recent studies of actin cytoskeletal dynamics in B cells were performed in the absence of LFA-1 ligation, we think our findings invite a critical “reset” for the way in which future B cell studies should be approached by highlighting the need for integrin co-stimulation when examining the roles of actin and myosin in B cell activation.

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4. 

   eLife

   Oct 5, 2021

   Evaluation Summary:

   This paper is of interest to immunologists studying mechanisms of lymphocyte activation and scientists in the broader field of cell mechanics. The work provides new insight into the cooperation among receptors, the actin cytoskeleton, and myosin motors that is required for the formation of a B cell immune synapse. The data support the key claims of the manuscript.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

   Read the original source
5. 

   eLife

   Oct 5, 2021

   Reviewer #1 (Public Review):

   A role for integrins in lowering the threshold for B cell activation was first observed over 15 years ago, but the mechanism has remained elusive. In this paper, Wang et al. investigate the role of LFA-1:ICAM-1 ligation in B cell synapse formation using live-cell super-resolution fluorescence microscopy in both primary B cells and the A20 B cell line. The use of super-resolution imaging is critical to the investigation as it reveals a level of organisation of the actomyosin network that is not visible with conventional microscopy approaches such as TIRF microscopy. They find that LFA-1:ICAM-1 ligation promotes the formation of actomyosin arcs that regulate various activities in the B cell synapse including BCR signalling, BCR:antigen microcluster transport, and the centralisation of antigen. In agreement …

   Reviewer #1 (Public Review):

   A role for integrins in lowering the threshold for B cell activation was first observed over 15 years ago, but the mechanism has remained elusive. In this paper, Wang et al. investigate the role of LFA-1:ICAM-1 ligation in B cell synapse formation using live-cell super-resolution fluorescence microscopy in both primary B cells and the A20 B cell line. The use of super-resolution imaging is critical to the investigation as it reveals a level of organisation of the actomyosin network that is not visible with conventional microscopy approaches such as TIRF microscopy. They find that LFA-1:ICAM-1 ligation promotes the formation of actomyosin arcs that regulate various activities in the B cell synapse including BCR signalling, BCR:antigen microcluster transport, and the centralisation of antigen. In agreement with earlier studies, they show that LFA-1:ICAM-1 ligation is required for B cells to centralise antigen that is present at very low density. They also demonstrate that myosin IIa contractility is required for the formation of the actomyosin arcs and promotes the exertion of strong traction forces on the antigen- and ICAM-1-presenting substrate. Using a small molecule inhibitor of formin activity in combination with miRNA knockdown of the formin mDia1, the authors show that the actomyosin arcs originate at the outer edge of the synapse and that their generation is formin dependent. These data provide a much-needed advance to our understanding of the role LFA-1 plays in the earliest events in B cell responses to antigen.

   The conclusions of the paper are mostly well supported by the data, but there are a few points that would need to be clarified.

   1. The requirement for LFA-1:ICAM-1 ligation in the formation of the actomyosin arcs is not clear. The authors observe that ~30% of B cells form actomyosin arcs with anti-IgM stimulation only (Figure 1). Does LFA-1:ICAM-1 ligation simply stabilise the arcs and therefore make their appearance more likely, or does it promote the formation of a distinct actomyosin network with unique functions? The images and videos selected to represent cells stimulated with anti-IgM only (Figure 1; Movies 1A and 1B) seem form a highly branched actin network throughout the synapse, but it would be informative to see cells having the actomyosin arcs for comparison. Since B cells stimulated with anti-IgM alone are capable of signalling and centralising antigen, it would be interesting to know whether and how these two populations (with and without arcs) differ.

   2. The authors propose that the contractile actomyosin network formed in the presence of LFA-1:ICAM-1 interactions promotes B cell activation especially at low antigen concentrations; however, their data focus only on early signalling (pCD79a and pCD19) and it would be helpful to know whether LFA-1:ICAM-1 interactions impact signalling further downstream.

   3. The observation that some GC B cells centralise antigen is very interesting, but there are a few aspects of this investigation that should be expanded upon. The authors show that with LFA-1:ICAM-1 interactions, GC B cells are about equally likely to organise BCR:antigen complexes into peripheral clusters and centralised clusters. It would be informative to have, in the same study (Figure 7), a comparison with GC B cells stimulated with antigen alone. The reason is that other studies investigating GC B cell synapse architecture did not quantify antigen organisation in this way, so it is difficult to make comparisons with previous work. It would also be very useful to see how the actomyosin network is organised in GC B cells exhibiting different synaptic architectures (i.e. peripheral versus central clusters), especially given the critical role of myosin IIa activity in GC B cell antigen affinity discrimination. Additionally, while it is a very interesting observation that LFA-1:ICAM-1 interactions may affect GC B cell synapse organisation, it is not clear whether this has an impact on cellular function. For instance, does antigen and actomyosin organisation in GC B cell synapses contribute to differences in signalling or traction force generation? In the introduction the authors suggest that actomyosin arcs contribute to antibody affinity maturation (line 87-88), but without functional studies to support this claim I think it is too speculative.

   Read the original source
6. 

   eLife

   Oct 5, 2021

   Reviewer #2 (Public Review):

   The manuscript utilizes elegant imaging tools to describe the contractile actomyosin arcs, induced by integrin-ligation, and their involvement in antigen gathering in B cells. The findings are important and have the potential to make a considerable impact in the field. The main conclusions are well supported by strong data and the manuscript convincingly brings across the need of integrin-ligation to induce generation of the arc network and the role of this structure in antigen gathering. The methods and the quality of imaging are state-of-the-art and provide an important example for future studies in B cell immune synapse. Some aspects of the study would benefit from clarification and extended experimentation or analysis.

   1. In addition to cultured B cells, the work includes naïve primary B cells as well as …

   Reviewer #2 (Public Review):

   The manuscript utilizes elegant imaging tools to describe the contractile actomyosin arcs, induced by integrin-ligation, and their involvement in antigen gathering in B cells. The findings are important and have the potential to make a considerable impact in the field. The main conclusions are well supported by strong data and the manuscript convincingly brings across the need of integrin-ligation to induce generation of the arc network and the role of this structure in antigen gathering. The methods and the quality of imaging are state-of-the-art and provide an important example for future studies in B cell immune synapse. Some aspects of the study would benefit from clarification and extended experimentation or analysis.

   1. In addition to cultured B cells, the work includes naïve primary B cells as well as isolated germinal center B cells. While the use of primary cells adds value to the study, in most cases the cells are activated first with LPS prior to transfection with F-Tractin constructs. Such a treatment is likely to alter the cytoskeletal features of the naïve B cells and, thus, it would be informative to provide an analysis of this effect.

   2. Various analyses in the manuscript rely on quantifying the different parts of the synapse, dSMAC, pSMAC and cSMAC. The regions are segmented manually and this is likely to be relatively ambiguous at times, especially in cells where the arc network is hampered. Yet, the segmentation has a direct and strong effect on the results, which is why this aspect would benefit from more emphasis.

   3. The example images are beautiful and detail robust arc organization, or lack of it in given conditions. However, it also seems likely that if only 70% of the cells have detectable arcs there are also a lot of cells with very poor arc content raising questions if a wider selection of representative images would be needed to evaluate the data. It would be important to describe the true variability of the cytoskeletal organization in the samples.

   4. The data on mDia1 would be strengthened by rescue-experiments or a scrambled sequence control.

   5. The authors show actomyosin arcs also in germinal center B cells data but while the functional role of arcs in antigen gathering or uptake is suggested it is not tested.

   Read the original source
7. 

   eLife

   Oct 5, 2021

   Reviewer #3 (Public Review):

   The work 'A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation' by Wang et al. describes the importance of integrin mediated B-cell co-stimulation for IS formation in B-cells by fostering the formation of myosin II A driven actin arcs that are essential in the transport of IgM clusters towards the IS center.

   The work presented here, i.e. experiments and analysis, is very thoroughly done and includes tests and controls using different labelling strategies and constructs of myosin II A, multiple cell types including primary cells and a range of chemical inhibitors to rule out artefacts.

   The authors claim that the observation of actin arcs in B-cells co-stimulated by ICAM-1 - LFA-1 interaction is important for the efficient activation of B-cells …

   Reviewer #3 (Public Review):

   The work 'A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation' by Wang et al. describes the importance of integrin mediated B-cell co-stimulation for IS formation in B-cells by fostering the formation of myosin II A driven actin arcs that are essential in the transport of IgM clusters towards the IS center.

   The work presented here, i.e. experiments and analysis, is very thoroughly done and includes tests and controls using different labelling strategies and constructs of myosin II A, multiple cell types including primary cells and a range of chemical inhibitors to rule out artefacts.

   The authors claim that the observation of actin arcs in B-cells co-stimulated by ICAM-1 - LFA-1 interaction is important for the efficient activation of B-cells in the presence of limiting levels of anti-IgM and this is very well supported by the experiments. However, it was a bit surprising that the paper did not draw much of parallels between the observed phenomenon and the reported actin arcs in activated T-cells even though some of the authors were very much involved in such work on T-cells. If there is a good reason to believe there is no ground to draw comparisons, this would then also need to be highlighted by the authors.

   The work on establishing the drivers of actin arc formation and dynamics is well done, but it is important to note that previous work has analyzed actin arc formation in other cell types. Work by Bershadsky has already established many 'ground rules' for the formation of actin arcs and the role of integrin adhesion, formin activity and myosin II in the process (Tee YH, Shemesh T, Thiagarajan V, Hariadi RF, Anderson KL, Page C, Volkmann N, Hanein D, Sivaramakrishnan S, Kozlov MM, Bershadsky AD. 2015. Cellular chirality arising from the self-organization of the actin cytoskeleton. Nat Cell Biol 17:445-457. doi:10.1038/ncb3137). It might be very instructive if the authors could put their findings in relation to this work.

   The formation of actin arcs is also well studied in U2OS cells and the results presented here could highlight interesting general features of this process observed in very different cell types (Tojkander S, Gateva G, Husain A, Krishnan R, Lappalainen P. 2015. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly. Elife 4:1-28. doi:10.7554/eLife.06126; Bur-nette DT, Shao L, Ott C, Pasapera AM, Fischer RS, Baird MA, Der Loughian C, Delanoe-Ayari H, Paszek MJ, Davidson MW, Betzig E, Lippincott-Schwartz J. 2014. A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells. J Cell Biol 205:83-96. doi:10.1083/jcb.201311104).

   In this regard, the findings about the importance of myosin II A activity, integrin adhesion and mDia1 in the formation of actin arcs is not that surprising and the authors might rather highlight the important role of these newly studied structures for co-stimulation in B-cells as this is the more novel and insightful bit of the work.

   Read the original source
8. 

   Version published to 10.1101/2021.08.12.456133v1 on bioRxiv

   Aug 13, 2021