The membrane-actin linkers ezrin, radixin, and moesin are dispensable for macrophage migration and cortex mechanics

  1. Perrine Verdys
  2. Javier Rey Barroso
  3. Joseph Vermeil
  4. Martin Bergert
  5. Thibaut Sanchez
  6. Arnaud Métais
  7. Thomas Mangeat
  8. Elisabeth Bellard
  9. Claire Bigot
  10. Jean-Philippe Girard
  11. Isabelle Maridonneau-Parini
  12. Christel Vérollet
  13. Frédéric Lagarrigue
  14. Alba Diz-Muñoz
  15. Julien Heuvingh
  16. Matthieu Piel
  17. Olivia Du Roure
  18. Véronique Le Cabec
  19. Sébastien Carréno
  20. Renaud Poincloux

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Abstract

The cellular actin cortex provides crucial mechanical support and plays critical roles in numerous functions, including cell division and migration. The proteins of the ERM family, ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the individual contributions of these three proteins to leukocyte migration, we generated single and triple ERM knock-out macrophages. Surprisingly, we found that even in the absence of ERMs, macrophages can still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore we discovered that, unlike every other cell type previously investigated, the single or triple knock-out of ERMs does not affect macrophage migration in a large diversity of contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanics of their actin cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their adaptive cortical plasticity.

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    General Statements

    Ezrin, Radixin, and Moesin (ERMs) serve as crucial cytoskeletal linker proteins, connecting the actin cytoskeleton to the plasma membrane upon activation. ERMs are essential regulators of cell morphogenesis across every cell types reported so far, and have been implicated in vital cellular functions such as migration, and invasion. In our study, we discovered that ERMs are dispensable for the cortical organization of macrophages. In accordance with this surprising finding, we found that the migration of macrophages was not affected upon knock-out of the three ERMs. Our findings challenge the prevailing belief that ERMs universally regulate cortical organization. Instead, they indicate that the actin cortex of macrophages has evolved to possess a high degree of adaptability and plasticity, enabling these immune cells to function independently of ERM proteins.

    We thank the editors of Review Commons that handled our manuscript and all three reviewers for their positive assessment of our manuscript and for their constructive suggestions.

    Description of the planned revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In the manuscript, the authors systematically test the role of ERM proteins in macrophages using RNA silencing as well as the genetic knockout approaches. Previous studies have highlighted the fundamental importance of ERM proteins as structural and regulatory components of the cell cortex governing several essential functions such as the generation of surface features such as filopodia, maintenance of cortex-plasma membrane attachment, bleb retraction, cortical mechanics, and cell migration. The authors performed a series of experiments to comprehensively test each of these functions (including cell migration in 2D surface and 3D matrix in vitro, ex vivo on tumor implants, as well as in vivo) and found that none of these are significantly affected when ERM proteins are downregulated in macrophages. Overall, the paper is solid, the experiments are well-designed and conclusive, and the manuscript is written well.

    We thank the reviewer for these encouraging comments.

    I have no significant concerns with the study. My only experimental suggestion is related to a previously shown function of ERM protein in macrophages- the ERM proteins play an important role in phagosome maturation in macrophages (Defacque et al., EMBO, 2000; Lars-Peter et al., PNAS, 2006; Mylvaganam et al., Current Biology, 2021). It would be nice if authors could explore this phenotype in their perturbation system.

    We thank the reviewer for this valuable suggestion. ERM proteins have indeed been proposed as important for macrophage phagocytosis. Importantly, their necessity for the early steps of this process is still debated, as conclusions differ depending on the cellular model used and the type of particle to be internalised (Erwig et al., PNAS USA 2006; Di Pietro et al., Sci. Rep. 2017; Gomez and Descoteaux, Biochem. Biophys. Res. Commun. 2018; Mu et al., Nat. Commun. 2018; Okazaki et al., J. Physiol. Sci. 2020). While the implication of ERMs in the early steps of phagocytosis remains controversial, there seems to be a consensus to implicate ezrin and moesin in phagosome maturation (Defacque et al. EMBO 2000 ; Erwig et al., PNAS USA 2006; Marion et al., Traffic 2011; Gomez and Descoteaux, Biochem. Biophys. Res. Commun. 2018).

    We have already started addressing the ability of ERM-depleted macrophages to perform phagocytosis. In particular, we quantified the dynamics of phagocytosis of ovalbumin-coated or IgG-opsonized polystyrene beads, which did not reveal any difference between WT and ERM-depleted macrophages.

    Proposed revision: We propose to include in the manuscript our quantification of IgG-coated and non-coated phagocytosis, and evaluate whether phago-lysosome fusion is delayed in ERM-depleted macrophages.

    A minor concern with the study is, as the authors have already pointed out, that ERM proteins may still be required for some functions in macrophages under specific (environmental?) conditions. It is of course impossible to experimentally test all possible conditions that may involve ERMs, however, the authors should include a note on the hypothetical conditions that may require ERMs in macrophages. They should also discuss possible hypothetical reasons why macrophages may have evolved a cortex that does not rely on ERM proteins for specific functions. Overall, a more extended discussion on the role of ERM proteins (or the lack of them) in macrophages is required.

    As suggested, in the revised version of the manuscript we will add a more extensive discussion of the role of ERM proteins in macrophages, and in particular the hypothetical conditions that might require their presence, as well as the reasons why macrophages have developed a particular cortex.

    Reviewer #1 (Significance (Required)):

    The manuscript is important on many accounts: The ERM proteins are considered crucial membrane-cytoskeletal linkers in many cellular systems. The study presents a surprising finding that cortical phenomena requiring membrane-cytoskeletal attachment do not essentially need ERM proteins providing a fundamental conceptual advance. The results from this study will also inform both experimental as well as theoretical studies of cortical organization and dynamics in the future. Furthermore, overexpressed mutant forms of ERMs are used as sensors as well as perturbing agents of cortical actin dynamics in many cellular systems. These utilities can now be further substantiated and if required, revised in light of the results from this study.

    I am an immune cell biologist specializing in early lymphocyte activation and cytoskeleton dynamics.

    We would like to thank the reviewer for pointing out the importance of our work for our understanding of the function of the cellular cortex, and for highlighting the fact that it may lead to a reinterpretation of the results obtained using ERM mutants.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary ERM proteins are known to play a central role in linking the cortical actin cytoskeleton with the plasma membrane, which is involved in regulating a diverse range of actin-rich membrane structures. The authors question the role that ERM proteins play in regulating cell shape changes and migration, specifically in macrophages. To test this, they designed an approach to systematically delete each ERM in macrophages - followed by the production of a triple-ERM ko line (tKO) using HoxB8 myeloid progenitor cells. The tKO line was subjected to a series of in vitro and in vivo experiments - all of which involve a series of imaging techniques to monitor membrane dynamics, protein subcellular organisation and cellular behaviours (e.g. rolling fraction, sticking fraction and chemotaxis). Their overall conclusion is that ERM are dispensable for macrophage membrane structures and migration.

    General comments.

    The experiments are very well executed. The manuscript in short demonstrates that the ERM proteins are dispensable for macrophage migration (both in 2D and 3D contexts), but there is very little beyond this work that points to what they might be doing instead. In this regard, given that the focus is exclusively on macrophage migration, the work comes across as quite specialised.

    We thank the reviewer for appreciating the quality of our work.

    We respectfully disagree to their assessment of the limited scope of our findings. Given the crucial importance of the migration of macrophages for so many of our body's functions, our findings will have a wide-ranging impact. Furthermore, and as acknowledged by Reviewers 1 and 3, we believe that the discovery that ERMs do not play a universal role in cortical mechanics and in cell migration, as hitherto believed, reaches a much wider scientific audience than that of the macrophage field. By proposing a unique research model (a triple KO for ERMs), our work allows to question many studies carried out with less direct molecular tools, such as the use of drugs or mutants of ERMs.

    We acknowledge the fact that although our data convincingly demonstrate that the ERM proteins are dispensable for macrophage migration, they do not reveal alternative functions for these proteins. We agree that it could be interesting to search for alternative functions for ERM proteins in macrophages in future studies. However, we believe that such studies are out of the scope of the present manuscript.

    The biggest concern I have is with the in vivo part. It should be noted that the work outlined in the manuscript does not actually address diapedesis, which is monitoring transmigration from the blood into tissue. Rolling and sticking do not define diapedesis. The experiments that the authors have conducted may have captured diapedesis events, but that very much depends on the length of time that the IVM was conducted. The authors would need to qualify their claims in this regard. Removing this work altogether would not lessen the impact, given that diapedesis is not shown. The work would therefore be very much in vitro/ex vivo.

    We agree with the reviewer that, due to technical limitations, we only measured the rolling and sticking capacity of the +/-ERM cells and did not measure diapedesis directly. Following Reviewer's comments, we have thus modified the text of the manuscript and no longer use the term ‘diapedesis’ to describe our in vivo intravital imaging studies.

    We also clarified the fact that we did not inject differentiated macrophages into the circulation, but macrophage precursors obtained by the treatment of progenitors with a 1 day treatment only (and not a 7 day treatment) with 20 ng/mL M-CSF.

    Here, highlighted in yellow, are the changes to the text (in the Introduction, Results, Methods and Legends sections):

    Introduction, p3:

    “Surprisingly, we found that ERMs are dispensable for macrophages to migrate in diverse contexts, including in vitro 2D migration and 3D invasion of extracellular matrix, ex vivo tissue infiltration through healthy dermis and tumor tissue, and for the in vivo adhesion of macrophage precursors to an activated endothelium.”

    Results, p6:

    “ERM tKo cells without ezrin, radixin, and moesin exhibit no impairment in their ability to adhere to vascular endothelium in vivo and infiltrate the ear derma or fibrosarcoma.

    To further investigate the migratory properties of ERM-deficient cells in vivo, we first assessed their ability to adhere to activated vascular endothelium into mice bearing a fibrosarcoma (Gui et al., 2018).”

    Results, p8:

    “Our study uncovered a surprising finding: ezrin, radixin and moesin are dispensable for key aspects of macrophage behavior, including the formation of lamellipodia and filopodia, the dynamics of membrane ruffles and podosomes, migration in vitro (in 2D or 3D matrices) and ex vivo (into dermis or tumor tissues) as well as for the in vivo adhesion of macrophage precursors to activated vascular endothelium).”

    Methods, p14:

    “In vivo analysis of adhesion to vascular endothelium with wide-field intravital microscopy”

    And

    “HoxB8-progenitors were directed towards monocyte/macrophage differentiation using a 1 day treatment with 20 ng/mL mouse M-CSF.”

    Figure 4 legend, p33:

    “Fig. 4: ERM tKO cells have no defect in adhesion to vascular endothelium in vivo and infiltrate tissues explants ex vivo

    1. In vivo adhesion to vascular endothelium

    Fibrosarcoma cells were injected into the flank of a mice. After a week, tumor was exposed for intravital microscopy, and the femoral artery of recipient mice was catheterized for injection of exogenous cells. Differentially labeled WT and TKO-ERM macrophage precursors were injected in the blood and their behaviour in tumor blood vessels was assessed by real-time imaging. Rolling fractions were quantified as the percentage of rolling cells in the total flux of cells in each blood vessel, and sticking fractions were quantified as the percentage of rolling cells that firmly adhered for a minimum of 30 seconds.”

    Proposed revision: We propose to keep the results of the in vivo experiments in the manuscript, including the modifications proposed by the reviewer and listed above.

    Specific questions

    How sure are the authors that they are capturing these events in cremasteric venules?

    As described in the Results and Methods section, these measurements were not captured in cremasteric venules but in fibrosarcoma tumour blood vessels, where we have previously demonstrated strong recruitment of circulating monocytes to infiltrate tumor tissue (Gui et al., Cancer Immunol. Res. 2018).

    Is there any sign of cells being trapped in the microcirculation?

    The diameter of the tumor blood vessels analysed is consistent with tumor post-capillary venules, and we have not seen cells trapped in these tumor blood vessels.

    The reason for injecting macrophages intravenously is not explained.

    We injected cells intravenously in order to compare their capacity to adhere to activated tumor blood vessels by intravital microscopy. This is now clarified in the corresponding result section (p6):

    “For that purpose, one day differentiated wild-type or ERM-deficient cells were fluorescently labelled with two different cell trackers, mixed in a 1:1 ratio, and co-injected intra-arterially into recipient mice in order to analyse their behaviour in tumor blood vessels by intravital microscopy.”

    Are these experiments modelling intravascular (patrolling) macrophages? Monocytes will typically differentiate into macrophages in tissue.

    We again apologize for the lack of clarity. In these experiments, we did not inject fully differentiated (seven days) macrophages but progenitors directed towards monocyte/macrophage differentiation using a 1 day treatment with 20 ng/mL mouse M-CSF. We believe that these experiments model the adhesion/recruitment of monocytes by activated vascular endothelium in the tumor microenvironment.

    The fact that the cells are able to "roll" and "stick" suggests that they have the complimentary cell adhesion molecules, although this is not addressed in the study.

    We agree with the reviewer. Our intravital microscopy analyses indicate that the injected cells have the complementary cell adhesion molecules for firm adhesion to activated tumor blood vessels. Importantly, our data clearly demonstrate that the capacity of ERM-tKO cells to bind vascular endothelium in the tumor microenvironment is similar to that of WT cells (Fig. 4A).

    Reviewer #2 (Significance (Required)):

    The strength of the manuscript is based on the robust in vitro experiments, however such experiments are difficult to address in vivo - mainly because of the issue that macrophages (unless patrolling macrophages) are not a useful model to investigate for ivm experiments.

    We thank the reviewer for recognizing the robustness of our in vitro experiments. We fully agree with the reviewer that the in vivo experiments are more challenging and that the behaviour of monocytes/(patrolling) macrophages is difficult to mimic in vivo. However, we believe that our intravital microscopy analyses are important because they demonstrate that ERM-tKO cells retain the capacity to bind firmly (sticking) to activated tumor blood vessels in vivo.

    This would be of great interest to the macrophage field, which is quite limited in scope. An advancement in the field would be to learn what is taking over the role of ERM in macrophages. As such, this becomes a report with a series of experiments to confirm that ERM are not involved.

    Again, we respectfully disagree with the reviewer, as this work goes against the dogma that ERMs are generally the most important mechanical links between the plasma membrane and the cytoskeleton. By clearly establishing that this is not the case in macrophages, cells whose importance for our immunity justifies the importance of their investigation, this study could make it possible to reconsider the functioning of the cellular cortex and the role of ERMs in other cellular systems.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Verdys and colleagues report an elegant study in which the authors describe that ERM proteins are dispensable for migrating monocyte-derived macrophages. The methods are adequate and the results support the conclusions.

    We thank the reviewer for these very supportive comments.

    Major points:

    - Although the authors demonstrate, by multiple methods, the dispensability of ERM proteins in the migration of macrophages derived from monocytes, the role of these proteins must also be evaluated in the phagocytosis process (another relevant functional aspect of macrophages).

    This is an excellent suggestion, which should make it possible to clarify the role of ERMs in this important function of macrophages.

    ERM proteins have indeed been proposed as important for macrophage phagocytosis. Importantly, their necessity for the early steps of this process is still debated, as conclusions differ depending on the cellular model used and the type of particle to be internalised (Erwig et al., PNAS USA 2006; Di Pietro et al., Sci. Rep. 2017; Gomez and Descoteaux, Biochem. Biophys. Res. Commun. 2018; Mu et al., Nat. Commun. 2018; Okazaki et al., J. Physiol. Sci. 2020). While the implication of ERMs in the early steps of phagocytosis remains controversial, there seems to be a consensus to implicate ezrin and moesin in phagosome maturation (Defacque et al. EMBO 2000 ; Erwig et al., PNAS USA 2006; Marion et al., Traffic 2011; Gomez and Descoteaux, Biochem. Biophys. Res. Commun. 2018).

    We have already started addressing the ability of ERM-depleted macrophages to perform phagocytosis. In particular, we quantified the dynamics of phagocytosis of ovalbumin-coated or IgG-opsonized polystyrene beads, which did not reveal any difference between WT and ERM-depleted macrophages.

    Proposed revision: We propose to include in the manuscript our quantification of IgG-coated and non-coated phagocytosis, and evaluate whether phago-lysosome fusion is delayed in ERM-depleted macrophages.

    • How is the activation of key downstream targets of ERM proteins involved in macrophage migration in KO models?_

    This is a very pertinent question. However, while ERMs have been described as being downstream of several signalling pathways, their own downstream targets are unfortunately poorly documented and, to our knowledge, none are known in macrophages.

    In different cellular contexts, it has been proposed that ERMs regulate PI3K (Gautreau et al. PNAS USA 1999), Ras (Sperka et al. Plos One 2011) or that they are involved in the initiation of protein translation (Briggs et al. Neoplasia 2012), but these results have not yet been confirmed and we believe they are outside the scope of this study.

    During macrophage migration, we consider that their obvious main target is cortical actin, and demonstrate in this manuscript that the functional coupling between actin and the plasma membrane is not affected by full ERM knockout.

    Reviewer #3 (Significance (Required)):

    Advance: The present study fills a gap in the participation of ERM proteins in cell migration. The results obtained on the dispensability of these proteins in macrophage migration can pave avenues for identifying new processes and proteins associated with migration in this context.

    Audience: The audience for this study is very broad.

    We again thank the reviewer for recognising the importance of this work for the understanding of cell migration.

    My expertise: I have expertise in cellular and molecular biology with a focus on processes associated with cancer. Among the numerous research fronts of the group led by me, we recently identified the EZR gene (which encodes the ezrin protein) as a prognostic marker and molecular target in acute leukemias.

    Description of the revisions that have already been incorporated in the transferred manuscript

    In the revised version of the article, we have taken into account all relevant changes proposed by the reviewers. We modified the text of the manuscript and no longer use the term ‘diapedesis’ to describe our in vivo intravital imaging studies, and clarified the fact that we did not inject differentiated macrophages into the circulation, but macrophage precursors obtained by the treatment of progenitors with a 1 day treatment only (and not a 7 day treatment) with 20 ng/mL M-CSF.

    Here, highlighted in yellow, are the changes to the text (in the Introduction, Results, Methods and Legends sections):

    Introduction, p3:

    “Surprisingly, we found that ERMs are dispensable for macrophages to migrate in diverse contexts, including in vitro 2D migration and 3D invasion of extracellular matrix, ex vivo tissue infiltration through healthy dermis and tumor tissue, and for the in vivo adhesion of macrophage precursors to an activated endothelium.”

    Results, p6:

    “ERM tKo cells without ezrin, radixin, and moesin exhibit no impairment in their ability to adhere to vascular endothelium in vivo and infiltrate the ear derma or fibrosarcoma.

    To further investigate the migratory properties of ERM-deficient cells in vivo, we first assessed their ability to adhere to activated vascular endothelium into mice bearing a fibrosarcoma (Gui et al., 2018). For that purpose, one day differentiated wild-type or ERM-deficient cells were fluorescently labelled with two different cell trackers, mixed in a 1:1 ratio, and co-injected intra-arterially into recipient mice in order to analyse their behaviour in tumor blood vessels by intravital microscopy.”

    Results, p8:

    “Our study uncovered a surprising finding: ezrin, radixin and moesin are dispensable for key aspects of macrophage behavior, including the formation of lamellipodia and filopodia, the dynamics of membrane ruffles and podosomes, migration in vitro (in 2D or 3D matrices) and ex vivo (into dermis or tumor tissues) as well as for the in vivo adhesion of macrophage precursors to activated vascular endothelium).”

    Methods, p14:

    “In vivo analysis of adhesion to vascular endothelium with wide-field intravital microscopy”

    And

    “HoxB8-progenitors were directed towards monocyte/macrophage differentiation using a 1 day treatment with 20 ng/mL mouse M-CSF.”

    Figure 4 legend, p33:

    “Fig. 4: ERM tKO cells have no defect in adhesion to vascular endothelium in vivo and infiltrate tissues explants ex vivo

    1. In vivo adhesion to vascular endothelium

    Fibrosarcoma cells were injected into the flank of a mice. After a week, tumor was exposed for intravital microscopy, and the femoral artery of recipient mice was catheterized for injection of exogenous cells. Differentially labeled WT and TKO-ERM macrophage precursors were injected in the blood and their behaviour in tumor blood vessels was assessed by real-time imaging. Rolling fractions were quantified as the percentage of rolling cells in the total flux of cells in each blood vessel, and sticking fractions were quantified as the percentage of rolling cells that firmly adhered for a minimum of 30 seconds.”

  6. Review Commons

    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

    Verdys and colleagues report an elegant study in which the authors describe that ERM proteins are dispensable for migrating monocyte-derived macrophages. The methods are adequate and the results support the conclusions.

    Major points:

    • Although the authors demonstrate, by multiple methods, the dispensability of ERM proteins in the migration of macrophages derived from monocytes, the role of these proteins must also be evaluated in the phagocytosis process (another relevant functional aspect of macrophages).
    • How is the activation of key downstream targets of ERM proteins involved in macrophage migration in KO models?

    Significance

    Advance: The present study fills a gap in the participation of ERM proteins in cell migration. The results obtained on the dispensability of these proteins in macrophage migration can pave avenues for identifying new processes and proteins associated with migration in this context.

    Audience: The audience for this study is very broad.

    My expertise: I have expertise in cellular and molecular biology with a focus on processes associated with cancer. Among the numerous research fronts of the group led by me, we recently identified the EZR gene (which encodes the ezrin protein) as a prognostic marker and molecular target in acute leukemias.

  7. Review Commons

    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

    ERM proteins are known to play a central role in linking the cortical actin cytoskeleton with the plasma membrane, which is involved in regulating a diverse range of actin-rich membrane structures. The authors question the role that ERM proteins play in regulating cell shape changes and migration, specifically in macrophages. To test this, they designed an approach to systematically delete each ERM in macrophages - followed by the production of a triple-ERM ko line (tKO) using HoxB8 myeloid progenitor cells. The tKO line was subjected to a series of in vitro and in vivo experiments - all of which involve a series of imaging techniques to monitor membrane dynamics, protein subcellular organisation and cellular behaviours (e.g. rolling fraction, sticking fraction and chemotaxis). Their overall conclusion is that ERM are dispensable for macrophage membrane structures and migration.

    General comments.

    The experiments are very well executed. The manuscript in short demonstrates that the ERM proteins are dispensable for macrophage migration (both in 2D and 3D contexts), but there is very little beyond this work that points to what they might be doing instead. In this regard, given that the focus is exclusively on macrophage migration, the work comes across as quite specialised.

    The biggest concern I have is with the in vivo part. It should be noted that the work outlined in the manuscript does not actually address diapedesis, which is monitoring transmigration from the blood into tissue. Rolling and sticking do not define diapedesis. The experiments that the authors have conducted may have captured diapedesis events, but that very much depends on the length of time that the IVM was conducted. The authors would need to qualify their claims in this regard. Removing this work altogether would not lessen the impact, given that diapedesis is not shown. The work would therefore be very much in vitro/ex vivo.

    Specific questions

    How sure are the authors that they are capturing these events in cremasteric venules? Is there any sign of cells being trapped in the microcirculation? The reason for injecting macrophages intravenously is not explained. Are these experiments modelling intravascular (patrolling) macrophages? Monocytes will typically differentiate into macrophages in tissue. The fact that the cells are able to "roll" and "stick" suggests that they have the complimentary cell adhesion molecules, although this is not addressed in the study.

    Significance

    The strength of the manuscript is based on the robust in vitro experiments, however such experiments are difficult to address in vivo - mainly because of the issue that macrophages (unless patrolling macrophages) are not a useful model to investigate for ivm experiments.

    This would be of great interest to the macrophage field, which is quite limited in scope.

    An advancement in the field would be to learn what is taking over the role of ERM in macrophages. As such, this becomes a report with a series of experiments to confirm that ERM are not involved.

  8. Review Commons

    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

    In the manuscript, the authors systematically test the role of ERM proteins in macrophages using RNA silencing as well as the genetic knockout approaches. Previous studies have highlighted the fundamental importance of ERM proteins as structural and regulatory components of the cell cortex governing several essential functions such as the generation of surface features such as filopodia, maintenance of cortex-plasmamembrane attachment, bleb retraction, cortical mechanics, and cell migration. The authors performed a series of experiments to comprehensively test each of these functions (including cell migration in 2D surface and 3D matrix in vitro, ex vivo on tumor implants, as well as in vivo) and found that none of these are significantly affected when ERM proteins are downregulated in macrophages. Overall, the paper is solid, the experiments are well-designed and conclusive, and the manuscript is written well.

    I have no significant concerns with the study. My only experimental suggestion is related to a previously shown function of ERM protein in macrophages- the ERM proteins play an important role in phagosome maturation in macrophages (Defacque et al., EMBO, 2000; Lars-Peter et al., PNAS, 2006; Mylvaganam et al., Current Biology, 2021). It would be nice if authors could explore this phenotype in their perturbation system.

    A minor concern with the study is, as the authors have already pointed out, that ERM proteins may still be required for some functions in macrophages under specific (environmental?) conditions. It is of course impossible to experimentally test all possible conditions that may involve ERMs, however, the authors should include a note on the hypothetical conditions that may require ERMs in macrophages. They should also discuss possible hypothetical reasons why macrophages may have evolved a cortex that does not rely on ERM proteins for specific functions. Overall, a more extended discussion on the role of ERM proteins (or the lack of them) in macrophages is required.

    Significance

    The manuscript is important on many accounts: The ERM proteins are considered crucial membrane-cytoskeletal linkers in many cellular systems. The study presents a surprising finding that cortical phenomena requiring membrane-cytoskeletal attachment do not essentially need ERM proteins providing a fundamental conceptual advance. The results from this study will also inform both experimental as well as theoretical studies of cortical organization and dynamics in the future. Furthermore, overexpressed mutant forms of ERMs are used as sensors as well as perturbing agents of cortical actin dynamics in many cellular systems. These utilities can now be further substantiated and if required, revised in light of the results from this study.

    I am an immune cell biologist specializing in early lymphocyte activation and cytoskeleton dynamics.

  9. Version published to 10.1101/2023.07.27.550674v1 on bioRxiv