Arp2/3 Complex Activity Enables Nuclear YAP for Naïve Pluripotency of Human Embryonic Stem Cells

  1. Nathaniel P. Meyer
  2. Tania Singh
  3. Matthew L. Kutys
  4. Todd Nystul
  5. Diane L. Barber

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Abstract

Our understanding of transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency that includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, uniform cell mechanics within naïve colonies, nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

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  1. Version published to 10.1101/2023.01.16.524285v3 on bioRxiv
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    Reply to the reviewers

    1. General Statements

    We would like to thank the reviewers for their professional comments and constructive suggestions. Our current plan is to revise the manuscript and supplemental materials in response to the reviewers’ requests and suggestions. Toward this goal we began experiments to obtain new data requested by the reviewers and anticipate the outlined experiments can be completed within the next three months.

    2. Description of the planned revisions:

    __Reviewer #1: __

    The results of experiments where Arp2/3 is blocked (Fig.2) should be confirmed by Arp2/3 knock-down and with an independent Arp2/3 inhibitor. Several are available (CK-869, Benproperine, Pimozide). For Fig.3 and 4, that would not be necessary, but to establish the specificity of the effect in fig.2 this is absolutely required.

    __Response: __As requested, we will include new data with CK-869, the indicated Arp2/3 complex inhibitor. We purchased the inhibitor and are currently confirming its efficacy before testing whether it inhibits transition to the hESC naïve state. However, we respectfully disagree with generating a Arp2/3 knock-down hESC line. Arp2/3 complex genes are known to be essential genes in both mouse and human embryonic stem cells (PMID: 29662178 and PMID: 31649057). Furthermore, reports on successful knockout of complex subunits indicate that additional genetic manipulations are needed to maintain cell survival, including knockout of INK4A/ARF to bypass apoptosis associated with Arp2 shRNA knockdown (PMID: 22385962) and genetic manipulations in mouse models (PMID: 22492726. Thus, knock-down of Arp2/3 complex members in our cells is beyond the scope of this manuscript.

    Yilmaz A, Peretz M, Aharony A, Sagi I, Benvenisty N. Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells. Nat Cell Biol. 2018 May;20(5):610-619. doi: 10.1038/s41556-018-0088-1. Epub 2018 Apr 16. PMID: 29662178.

    Shohat S, Shifman S. Genes essential for embryonic stem cells are associated with neurodevelopmental disorders. Genome Res. 2019 Nov;29(11):1910-1918. doi: 10.1101/gr.250019.119. Epub 2019 Oct 24. PMID: 31649057; PMCID: PMC6836742.

    Wu C, Asokan SB, Berginski ME, Haynes EM, Sharpless NE, Griffith JD, Gomez SM, Bear JE. Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis. Cell. 2012 Mar 2;148(5):973-87. doi: 10.1016/j.cell.2011.12.034. PMID: 22385962; PMCID: PMC3707508.

    I believe that the status of the actin cytoskeleton in both states is not well enough characterized. This is especially obvious for branched actin networks themselves that depend on the Arp2/3. To this end, the authors may localize Arp2/3 or cortactin, a useful surrogate that often gives a better staining. This point is particularly important since contractile fibers are not made of branched actin. Myosin cannot walk or pull along branched actin networks because of steric hindrance. It might well be that branched actin networks are debranched after Arp2/3 polymerization. I suggest staining tropomyosins that would indicate where the transition between branched and unbranched actin would be. Along this line, phosphoERMs should be localized and revealed by Western blots (we expect an increase from primed to naive state) because they cannot perform the proposed function of linker between the membrane and actin filaments if they are not phosphorylated.

    Response: As requested we will include new data with cortactin immunolabeling, which we already completed. These new data, shown below, confirm that cortactin, which binds to branched actin filaments, co-localizes with the F-actin fence around hESC naïve colonies, suggesting that the fence includes branched F-actin. Also as requested, we are currently immunoblotting for phosphorylated ERMs to more thoroughly assess if they may serve as a linker between the membrane and actin filaments.

    Branched actin is required for cell cycle progression and cell proliferation in normal cells. This requirement is lost in most cancer cells (Wu et al., Cell 2012; Molinie et al., Cell Res 2019). This would be really important to know whether ESCs stop proliferating upon CK-666 treatment. In other words, do they behave like normal cells or transformed cells. Proliferation is a major function that depends on the YAP pathway. Cell counts and EdU incorporation can easily provide answers to this important question.

    __Response: __As requested, we will include new data on proliferation. We anticipate that these new data will complement data we already have showing that CK-666 does not impair proliferation compared with hESC controls. We also note that the role of the actin cytoskeleton in proliferation is well established and an increase in proliferation is a hallmark of acquisition of the naïve state of pluripotency (PMID: 35005567).

    Chen C, Zhang X, Wang Y, Chen X, Chen W, Dan S, She S, Hu W, Dai J, Hu J, Cao Q, Liu Q, Huang Y, Qin B, Kang B, Wang YJ. Translational and post-translational control of human naïve versus primed pluripotency. iScience. 2021 Dec 17;25(1):103645. doi: 10.1016/j.isci.2021.103645. PMID: 35005567; PMCID: PMC8718978.

    Minor Comments:

    What about the rescue of cell morphology? Does active YAP restore the intercellular contractile bundle?

    __Response: __As requested, we obtained these data, as shown below. Expression of the YAP-S127A mutant does rescue the formation of the actin ring architecture in the presence of CK666. We are currently performing additional dedifferentiation assays to immunolabel for pMLC and address the question of if expression of YAP-S127A restores the contractile bundle.

    __Reviewer #2: __

    The authors found that a ring of actin filaments at the colony periphery was characteristic of the naive hESCs. However, because all the data are presented as an image of a single confocal section, the 3D organization of the actin filaments is not clear. Although the authors drew a scheme for this actin ring being located in the apical domain of polarized cells, such data have not been provided in the manuscript. Since naive hESCs form dome-like colonies, it is important to show the 3D organization of actin filaments in the colony. 3D reconstruction of confocal microscopy images of the naive hESC colonies is required to show the relationship between actin filaments, adherens junctions, and the nuclei (as a reference for the Z axis). If 3D reconstruction is not technically possible, confocal images at different Z levels and maximum projection images should be obtained and provided.

    __Response: __As requested, we are currently generating 3D images of the actin fence by using Imaris software, which we previously used to show 3D images of mitochondrial morphology (PMID: 34038242)

    Manoli SS, Kisor K, Webb BA, Barber DL. Ethyl isopropyl amiloride decreases oxidative phosphorylation and increases mitochondrial fusion in clonal untransformed and cancer cells. Am J Physiol Cell Physiol. 2021 Jul 1;321(1):C147-C157. doi: 10.1152/ajpcell.00001.2021. Epub 2021 May 26. PMID: 34038242; PMCID: PMC8321791.

    Some of the statistical analyses were inappropriate. The authors have used Student's t-test for all analyses; however, one-way ANOVA and post-hoc analysis must be used to compare three or more groups (Figs. 2B, D, E, 3G, 4B, D, E).

    __Response: __As requested, we will re-evaluate our statistical analysis. We note that our submission reports comparisons between two groups, and hence, Student’s t-test is appropriate. For example, we compared primed and naïve to demonstrate successful acquisition of naïve pluripotency, and then we compared the naïve condition to the CK666-treated conditions to demonstrate the impact of CK666-treatment. As Reviewer 2 suggests we will reanalyze all quantifications using one-way ANOVA with post-hoc analysis in the full revision and we will also discuss with Stuart Gansky, a statistician at UCSF whom we previously consulted for most appropriate statistical analysis of our studies.

    Minor Comments:

    Page 9, second paragraph. In the discussion section, authors have written that "Cells within the ICM of mouse blastocysts exclude YAP from the nucleus whereas cells within the ICM of human blastocysts maintain nuclear YAP." However, a recent study has reported that the ICM/epiblast of mouse late blastocysts also express nuclear YAP. Epiblast Formation by TEAD-YAP-Dependent Expression of Pluripotency Factors and Competitive Elimination of Unspecified Cells. Hashimoto M, Sasaki H. Dev Cell. 2019, 50:139-154.e5. doi: 10.1016/j.devcel.2019.05.024.

    __Response: __As requested, we will revise our Discussion section to include findings from the indicated new publication.

    Reviewer #3:

    Many of their conclusions seem to be based on the qualitative analysis of a single image (e.g. Figures 1D-G, Fig 2G, Supplementary Figure 2). The authors should provide quantitative information regarding these analyses and indicate the number of cells/replicas collected for each experiment.

    __Response: __As requested, our revision will have added quantitative data when feasible. We note that in the field, traction force microscopy isn’t commonly quantified beyond including scale bars, which our original manuscript shows. Moreover, pluripotency is standardly not quantified because it is a binary switch - cells are either double positive or they are not. We show 100% double positive, and rtPCR data with known stage-specific markers.

    The actin ring surrounding hESCs colonies was previously described by Närvä et al. Although the authors cited this previous work, they do not discuss in deep the differences and similarities with their observations.

    __Response: __As requested, our revised manuscript with include additional detail comparing our results with those from Närvä et al. In brief, we observe the formation of this actin ring only in the naïve state of pluripotency, whereas Närvä et al. observe an actin architecture in the primed state. One possible source of difference between their study and ours are the cells used for analysis. Närvä et al. utilize induced pluripotent stem cells, long since proposed to be closer to naïve pluripotency than primed stem cells as conventionally isolated and maintained (see PMID: 27424783 and PMID: 19497275). Additionally, we observe that the contractile actin ring in naïve pluripotent stem cells is in a higher z-plane than reported by Närvä et al., although a direct comparison is difficult to make.

    Theunissen TW, Friedli M, He Y, Planet E, O'Neil RC, Markoulaki S, Pontis J, Wang H, Iouranova A, Imbeault M, Duc J, Cohen MA, Wert KJ, Castanon R, Zhang Z, Huang Y, Nery JR, Drotar J, Lungjangwa T, Trono D, Ecker JR, Jaenisch R. Molecular Criteria for Defining the Naive Human Pluripotent State. Cell Stem Cell. 2016 Oct 6;19(4):502-515. doi: 10.1016/j.stem.2016.06.011. Epub 2016 Jul 14. PMID: 27424783; PMCID: PMC5065525.

    Nichols J, Smith A. Naive and primed pluripotent states. Cell Stem Cell. 2009 Jun 5;4(6):487-92. doi: 10.1016/j.stem.2009.05.015. PMID: 19497275.

    The qualitative observation of Figure 3F suggests a lower overall YAP levels in primed and +CK666 cells in comparison to naive cells. Could the authors check if this is correct and, if this is the case, explain the observation?

    __Response: __As requested, our revision will include new data on YAP protein expression by immunoblotting.

    The authors should discuss deeper the rationale of the pan-ERM immunostaining experiments (since they used the individual antibodies afterwards) and provide a brief discussion of their results and, in particular, the colocalization with moesin but not with ezrin or radixin.

    __Response: __As requested, our revised manuscript will include a more detailed discussion of our results with ERM immunolabeling.

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

    __Reviewer #1: __

    Minor Comments:

    Fig2F: non-representative pictures or wrong quantification of the CK666 condition.

    __Response: __We thank the review for alerting us to this error. The CK666 Primed and Naïve condition images were swapped. We have edited the figure to correct this.

    Fig3A: Y-axis? What is it? How is it adjusted? -Log P?

    __Response: __Please see the methods section. Differential expression analysis was performed using DESeq2 R package. The resulting P values were adjusted (padj) using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with a padj

    Colors of dots not really visible (in reference to Figure 3A).

    __Response: __We thank the reviewer for this comment and have updated the figure to use more standard, colorblind-friendly color choices (see the above figure). Additionally, we fixed a drawing error in the figures when creating the volcano plots.

    Typos: Apr2/3 in the abstract, Hoeschst in Fig.S1B.

    __Response: __We thank the review for alerting us to these errors. We have edited the manuscript to correct them.

    __Reviewer #3: __

    There are many experimental details missing that are extremely relevant to fully understand the experiments and evaluate the robustness of the analyses (e.g., microscopy setup, fluorescent probes used for immunostaining, incubation conditions with the inhibitors SMIFH2 and CK666).

    __Response: __As requested we have updated the Materials and Methods section with more detailed information on procedures and reagents.

    Minor Comments:

    The Introduction makes the reader think that actin is the only cytoskeletal network involved in embryo development and stem cell properties. They should also include a brief discussion on the relevance of the other cytoskeletal networks in mechanotransduction and cell fate decisions.

    __Response: __As requested, we will revise our Introduction. We note, however, that in the field additional cytoskeleton components, including intermediate filaments and microtubules have mostly been shown for interacting with the nucleus with limited evidence for roles in differentiation.

    Many of the images seem to require a flat-field correction. Could the authors check that the illumination is homogeneous? This artifact could affect the data analysis.

    __Response: __As we indicate in the Methods section, the spinning disc confocal microscopes used in our study are equipped with a Borealis to mitigate uneven illumination across the field of view. Additionally, quantification in Figures 2C-E, Figures 3F-G, and Figures 4A-D are comparing measurements to a local background (i.e. cytoplasm nearby) in order to normalize for any uneven illumination effects.

    There are many abbreviations that are not defined in the text and are extremely specific to the field.

    __Response: __As requested, we have expanded the definition of many abbreviations in the text and any additional abbreviations changes will be clearly defined in our revised manuscript.

    Could the authors explain the selection of the pluripotency markers studied by qPCR? Specifically, why they studied DNMT3L, DPPA3, KLF2, and KLF4 (Fig. 1B) and the different set PECAM1, ESRRB, KLF4, and DNMT3L in Fig. 2B.

    __Response: __Defining the exact molecular and cell behavioral characteristics of naïve pluripotency remains an evolving point of development within the field. The pluripotency markers used in both original panels are known and established markers of naïve pluripotency. The original panel of DNMT3L, DPPA3, KLF2, and KLF4 was established based upon RNAseq datasets publicly available, whereas the secondary panel of PECAM1, ESRRB, KLF4, and DNMT3L was a more targeted analysis of genes found in the literature which have been interrogated in more detail for potential roles in naïve pluripotency. To facilitate clarity within the manuscript, we have updated Fig. 1B to match Fig. 2B for the purposes of defining a transcriptional hallmark of naïve pluripotency for the purposes of this manuscript.

    Figures 1G and 2G, please include the images of the colonies.

    __Response: __As requested, our revised manuscript will include phase contrast images, which we already have, as shown below. These images will be included Supplemental Figure 1 and Supplemental Figure 2 for the colonies used to show representative tractions in Figure 1G and 2G, respectively.

    3. Description of analyses that authors prefer not to carry out

    Reviewer #1:

    The results of experiments where Arp2/3 is blocked (Fig.2) should be confirmed by Arp2/3 knock-down and with an independent Arp2/3 inhibitor. Several are available (CK-869, Benproperine, Pimozide). For Fig.3 and 4, that would not be necessary, but to establish the specificity of the effect in fig.2 this is absolutely required.

    __Response: __As requested, we will include new data with CK-869, the indicated Arp2/3 complex inhibitor. We purchased the inhibitor and are currently confirming its efficacy before testing whether it inhibits transition to the hESC naïve state. However, we respectfully disagree with generating a Arp2/3 knock-down hESC line. Arp2/3 complex genes are known to be essential genes in both mouse and human embryonic stem cells (PMID: 29662178 and PMID: 31649057). Furthermore, reports on successful knockout of complex subunits indicate that additional genetic manipulations are needed to maintain cell survival, including knockout of INK4A/ARF to bypass apoptosis associated with Arp2 shRNA knockdown (PMID: 22385962) and genetic manipulations in mouse models (PMID: 22492726. Thus, knock-down of Arp2/3 complex members in our cells is beyond the scope of this manuscript.

    Yilmaz A, Peretz M, Aharony A, Sagi I, Benvenisty N. Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells. Nat Cell Biol. 2018 May;20(5):610-619. doi: 10.1038/s41556-018-0088-1. Epub 2018 Apr 16. PMID: 29662178.

    Shohat S, Shifman S. Genes essential for embryonic stem cells are associated with neurodevelopmental disorders. Genome Res. 2019 Nov;29(11):1910-1918. doi: 10.1101/gr.250019.119. Epub 2019 Oct 24. PMID: 31649057; PMCID: PMC6836742.

    Wu C, Asokan SB, Berginski ME, Haynes EM, Sharpless NE, Griffith JD, Gomez SM, Bear JE. Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis. Cell. 2012 Mar 2;148(5):973-87. doi: 10.1016/j.cell.2011.12.034. PMID: 22385962; PMCID: PMC3707508.

  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript entitled "Arp2/3 Complex Activity Enables Nuclear YAP for Naïve Pluripotency of Human Embryonic Stem Cells" by Nathaniel Meyer, Tania Singh, Matthew Kutys, Todd Nystul, and Diane Barber analyzes the formation of the actin ring that surrounds naive but not primed colonies of human embryonic stem cells (hESCs). The authors claim that the formation of this actin ring requires Arp2/3 which also modulates YAP localization. Despite the overall topic is relevant to understand key aspect of stem cells and embryo development, I have found several flaws in the manuscript (stated below) that, in my opinion, prevent its publication in its current form:

    1. Many of their conclusions seem to be based on the qualitative analysis of a single image (e.g. Figures 1D-G, Fig 2G, Supplementary Figure 2). The authors should provide quantitative information regarding these analyses and indicate the number of cells/replicas collected for each experiment.
    2. Many of the images seem to require a flat-field correction. Could the authors check that the illumination is homogeneous? This artifact could affect the data analysis.
    3. The actin ring surrounding hESCs colonies was previously described by Närvä et al. Although the authors cited this previous work, they do not discuss in deep the differences and similarities with their observations.
    4. There are many experimental details missing that are extremely relevant to fully understand the experiments and evaluate the robustness of the analyses (e.g., microscopy setup, fluorescent probes used for immunostaining, incubation conditions with the inhibitors SMIFH2 and CK666).
    5. The qualitative observation of Figure 3F suggests a lower overall YAP levels in primed and +CK666 cells in comparison to naive cells. Could the authors check if this is correct and, if this is the case, explain the observation?
    6. The authors should discuss deeper the rationale of the pan-ERM immunostaining experiments (since they used the individual antibodies afterwards) and provide a brief discussion of their results and, in particular, the colocalization with moesin but not with ezrin or radixin.

    Minor observations:

    1. The Introduction makes the reader think that actin is the only cytoskeletal network involved in embryo development and stem cell properties. They should also include a brief discussion on the relevance of the other cytoskeletal networks in mechanotransduction and cell fate decisions.
    2. There are many abbreviations that are not defined in the text and are extremely specific to the field.
    3. Could the authors explain the selection of the pluripotency markers studied by qPCR? Specifically, why they studied DNMT3L, DPPA3, KLF2, and KLF4 (Fig. 1B) and the different set PECAM1, ESRRB, KLF4, and DNMT3L in Fig. 2B.
    4. Figures 1G and 2G, please include the images of the colonies.

    Significance

    The manuscript analyzes the formation of the actin ring that surrounds naive but not primed colonies of human embryonic stem cells (hESCs). The authors claim that the formation of this actin ring requires Arp2/3 which also modulates YAP localization. Despite the overall topic is relevant to understand key aspect of stem cells and embryo development, I have found several flaws in the manuscript (stated below) that, in my opinion, prevent its publication in its current form (see Evidence, reproducibility and clarity)

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    This paper describes the involvement of the actin regulator Arp2/3 in the dedifferentiation of primed human embryonic stem cells (hESCs) into naive pluripotency. The authors initially demonstrated a reorganization of the actin cytoskeleton during the transition to naive pluripotency, which included the formation of a contractile actin ring at the colony periphery. Actin reorganization was also associated with a reduction in cell-substrate traction forces and an increase in cell-cell junction traction forces. The authors showed that the activity of the Arp2/3 complex was required for actin reorganization and acquisition of a naive pluripotent state. RNA-seq analysis revealed that the Arp2/3 complex regulates Hippo signaling. Furthermore, inhibition of Arp2/3 suppressed the nuclear localization of YAP, and expression of nuclear-localized YAP restored naive dedifferentiation of the Arp2/3 inhibited hESCs. Based on these results, the authors have proposed a model in which naive pluripotency is characterized by Arp2/3 complex-dependent remodeling of the actin cytoskeleton and colony mechanics. Additionally, it has been suggested that Arp2/3 activity facilitates naive dedifferentiation by promoting the nuclear translocation of YAP.

    Major comments:

    The experiments were of high quality, and the paper was clearly written with these major conclusions being supported by the experiments. However, the three-dimensional (3D) organization of F-actin, including the actin ring surrounding naive colonies, is unclear.

    1. The authors found that a ring of actin filaments at the colony periphery was characteristic of the naive hESCs. However, because all the data are presented as an image of a single confocal section, the 3D organization of the actin filaments is not clear. Although the authors drew a scheme for this actin ring being located in the apical domain of polarized cells, such data have not been provided in the manuscript. Since naive hESCs form dome-like colonies, it is important to show the 3D organization of actin filaments in the colony. 3D reconstruction of confocal microscopy images of the naive hESC colonies is required to show the relationship between actin filaments, adherens junctions, and the nuclei (as a reference for the Z axis). If 3D reconstruction is not technically possible, confocal images at different Z levels and maximum projection images should be obtained and provided.
    2. Some of the statistical analyses were inappropriate. The authors have used Student's t-test for all analyses,; however, one-way ANOVA and post-hoc analysis must be used to compare three or more groups (Figs. 2B, D, E, 3G, 4B, D, E).

    Minor comments:

    1. Page 9, second paragraph. In the discussion section, authors have written that "Cells within the ICM of mouse blastocysts exclude YAP from the nucleus whereas cells within the ICM of human blastocysts maintain nuclear YAP." However, a recent study has reported that the ICM/epiblast of mouse late blastocysts also express nuclear YAP.

    Epiblast Formation by TEAD-YAP-Dependent Expression of Pluripotency Factors and Competitive Elimination of Unspecified Cells. Hashimoto M, Sasaki H. Dev Cell. 2019, 50:139-154.e5. doi: 10.1016/j.devcel.2019.05.024.

    Significance

    The importance of actin dynamics and cell mechanics as regulators of cell fate transition has been demonstrated in several systems, including the differentiation of hESCs (ref 54). The importance of YAP in the generation of naive hESCs has been reported previously (24). This study further extends this knowledge by showing the importance of actin dynamics and cell mechanics during the naive dedifferentiation process of hESCs. Although the advancement is not significant, the identification of the Arp2/3 complex as an essential upstream regulator of actin dynamics, cell mechanics, and YAP provides novel and important information for the field of stem cell biology, specifically for researchers working on hESC reprogramming and regenerative medicine.

    The field of expertise of this reviewer is mouse preimplantation development and Hippo signaling.

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript by Meyer et al., shows that the transition from primed to naïve human Embryonic Stem cells is associated with changes with the organization of the actin cytoskeleton, mechanics exerted on the substratum and YAP activity. These changes require Arp2/3 activity and if these changes are blocked with an Arp2/3 inhibitor, the phenotype can be rescued by the expression of a constitutively active YAP form.

    This brief manuscript is overall well written and presented. The results are quite original, since branched actin polymerized by Arp2/3 is generally associated with membrane protrusions, and not with contractile actin fibers, as described here. Similarly, YAP activation has been shown to be regulated by RhoA-mediated contractility and here seems to depend on branched actin networks. I have nothing against these provocative conclusions, but I believe that to make their point stronger, more than just the use of an Arp2/3 inhibitor is required.

    Major Comments

    1. The results of experiments where Arp2/3 is blocked (Fig.2) should be confirmed by Arp2/3 knock-down and with an independent Arp2/3 inhibitor. Several are available (CK-869, Benproperine, Pimozide). For Fig.3 and 4, that would not be necessary, but to establish the specificity of the effect in fig.2 this is absolutely required.
    2. I believe that the status of the actin cytoskeleton in both states is not well enough characterized. This is especially obvious for branched actin networks themselves that depend on the Arp2/3. To this end, the authors may localize Arp2/3 or cortactin, a useful surrogate that often gives a better staining. This point is particularly important since contractile fibers are not made of branched actin. Myosin cannot walk or pull along branched actin networks because of steric hindrance. It might well be that branched actin networks are debranched after Arp2/3 polymerization. I suggest staining tropomyosins that would indicate where the transition between branched and unbranched actin would be. Along this line, phosphoERMs should be localized and revealed by Western blots (we expect an increase from primed to naive state) because they cannot perform the proposed function of linker between the membrane and actin filaments if they are not phosphorylated.
    3. Branched actin is required for cell cycle progression and cell proliferation in normal cells. This requirement is lost in most cancer cells (Wu et al., Cell 2012; Molinie et al., Cell Res 2019). This would be really important to know whether ESCs stop proliferating upon CK-666 treatment. In other words, do they behave like normal cells or transformed cells. Proliferation is a major function that depends on the YAP pathway. Cell counts and EdU incorporation can easily provide answers to this important question.

    Minor comments

    1. Fig2F: non-representative pictures or wrong quantification of the CK666 condition.
    2. Fig3A: Y-axis ? What is it ? How is it adjusted ? -Log P ?
    3. Colors of dots not really visible.
    4. What about the rescue of cell morphology ? Does active YAP restore the intercellular contractile bundle ?
    5. Typos: Apr2/3 in the abstract, Hoeschst in Fig.S1B.

    Significance

    This brief report would be a strong report if the major points are addressed. The conclusions are original, with a role of branched actin in inducing an intercellular contractile bundle and activating YAP, and important for a cell system of importance, human ESC. It would interest a wide variety of readers with either an interest in the actin cytoskeleton or in stem cells.

    I believe that the time required to address these 3 points is reasonable in the order of 3 months only.

    My expertise is the actin cytoskeleton.

  6. Version published to 10.1101/2023.01.16.524285v2 on bioRxiv
  7. Version published to 10.1101/2023.01.16.524285v1 on bioRxiv