Gαi2 Interaction with EB1 Controls Microtubule Dynamics and Rac1 Activity in Xenopus Neural Crest Cell Migration

  1. Soraya Villaseca
  2. Juan Ignacio Leal
  3. Lina Mariana Tovar
  4. María José Ruiz
  5. Jossef Guajardo
  6. Hernan Morales-Navarrete
  7. Roberto Mayor
  8. Marcela Torrejón

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Abstract

Cell migration is a complex and essential process in various biological contexts, from embryonic development to tissue repair and cancer metastasis. Central to this process are the actin and tubulin cytoskeletons, which control cell morphology, polarity, focal adhesion dynamics, and overall motility in response to diverse chemical and mechanical cues. Despite the well-established involvement of heterotrimeric G proteins in cell migration, the precise underlying mechanism remains elusive, particularly in the context of development. This study explores the involvement of Gαi2, a subunit of heterotrimeric G proteins, in cranial neural crest cell migration, a critical event in embryonic development. Our research uncovers the intricate mechanisms underlying Gαi2 influence, revealing a direct interaction with the microtubule-associated protein EB1, and through this with tubulin, suggesting a regulatory function in microtubule dynamics modulation. Here, we show that Gαi2 knockdown leads to microtubule stabilization, alterations in cell polarity and morphology with an increased Rac1-GTP concentration at the leading edge and cell-cell contacts, impaired cortical actin localization and focal adhesion disassembly. Interestingly, in Gαi2 knockdown cells, RhoA-GTP was found to be reduced at cell-cell contacts and concentrated at the leading edge, providing evidence of Gαi2 significant role in polarity. Remarkably, treatment with nocodazole, a microtubule-depolymerizing agent, effectively reduces Rac1 activity, restoring cranial NC cell morphology, actin distribution, and overall migration. Collectively, our findings shed light on the intricate molecular mechanisms underlying cranial neural crest cell migration and highlight the pivotal role of Gαi2 in orchestrating microtubule dynamics through EB1 and EB3 interaction, modulating Rac1 activity during this crucial developmental process.

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    Response to reviewer comments

    R: We really appreciate the reviewer positive comments and consideration, and we believe that the review process has significantly strengthened our manuscript.

    We have responded to all the reviewer comments, as follows:

    Response (R)

    FROM REVIEWER #1

    Major comments:

    The manuscript is mostly well written (it could use a few minor grammatical corrections), the significance of the problem is well described, and the results are clearly presented with adequate controls. The movies, provided as supplementary material, are of the highest quality and are essential additions to the stills provided in the figures. The data convincingly support the key conclusions of the manuscript.

    R: We sincerely appreciate the positive comments provided by the reviewer. In response, we have thoroughly revised the manuscript to address any grammatical issue.

    Does the MO knockdown both S and L homeologs of X. laevis? Since the level of GAPDH in Figure 1H also looks reduced in Gai2 MO lane, it should be made clear that the apparent knockdown of Gai2 was normalized to GAPDH, rather than being the results of unequal loading of the gel. Yes, I recognize that Figure 1I says normalized, but this is not stated in the results or the methods. Also, was this experiment done with X. laevis or X. tropicalis? I could imagine that if done in X. laevis, the lack of complete knockdown might be due to only one homeolog being affected.

    R: We appreciate the reviewer comment, and we described in Material and Methods section the region targeted by the morpholino, in both Xenopus species. We added the next paragraph in the Material and Methods section, see page 24, paragraph 2, lines: 4-11:

    "MO against Xenopus Gαi2 was designed by GeneTools to target the 5' UTR site of X. tropicalis (X.t) and X. laevis (X.l) transcripts (Gαi2MO: 5'-CGACACAGCCCCAGATAGTGCGT-3'). Specifically, it hybridizes with the 5' UTR of X. t Gαi2 (NM_203919), 17 nucleotides upstream of the ATG start codon. For X. l Gαi2, the morpholino hybridizes with both isoforms described in Xenbase. It specifically targets the 5' UTR of the Gαi2.L isoform (XM_018258962), located 17 nucleotides upstream of the ATG start codon, and the 5' UTR of the Gαi2.S isoform (NM_001097056), situated 275 nucleotides upstream of the ATG."

    With respect to Figure 1H and 1I, we have specified in the Fig. 1 legend that we normalized the data to GAPDH to quantifying the decrease in Gαi2 expression induced by the morpholino.

    See page 40, Figure 1H-I, Legends section. Finally, the result showed in Fig. 1A-I was done in X.t., that was now stated at the legend from the figure. We added at the Supplementary material Fig.1S, the result done in X.l. experiment.

    The knowledge of the efficacy of knockdown in each Xenopus species provided by the information requested in the previous point, would allow the reader to assess the level of knockdown in the remaining assays. To do this, the authors should tell us which assays were done in which species. I am not suggesting that each experiment needs to be done in each species, only that the information should be provided. If the MO is more effective in X. tropicalis - which assays used this species? If the knock down is partial, as shown in Figure 1H-I, which species this represents in the remaining assays would be useful knowledge.

    R: We greatly appreciate the reviewer's valuable comments and suggestions, and as a response, we have incorporated a new supplementary figure (Figure S1). This figure includes a western blot and an in situ hybridization assay illustrating the efficiency of the knockdown in Xenopus laevis. The results presented in Figure S1 demonstrate that the knockdown efficiency is similar in both Xenopus species, allowing for a comparison between Figure 1A-I (X. tropicalis) and Figure 1S (X. laevis).

    To complement this information, we have also improved the section of Material and Methods regarding the experiments in both Xenopus species (Xenopus tropicalis and Xenopus laevis). As detailed in the Materials and Methods section, we employed 20 ng of Gai2MO for Xenopus tropicalis embryos and 35 ng of Gai2MO for Xenopus laevis embryos to deplete cell migration. In both species, in vivo migration was analyzed, resulting in a substantial inhibition of cranial neural crest (NC) migration, ranging from 60% to 80%. Additionally, we conducted dispersion assays in both species. In X. laevis, in vitro migration was monitored for 10 hours, while in X. tropicalis, it was tracked for 4 hours, both yielding the same phenotype. We also studied cell morphology and microtubule dynamics in both Xenopus models. However, we used different tracer concentrations for each, with 200 pg for X. laevis and 100 pg for X. tropicalis, as specified in the Materials and Methods section. Our Rac1 and RhoA timelapse experiments were conducted in both species as well, employing pGBD-GFP and rGBD-mCherry probes, respectively, and different probe concentrations as outlined in the Materials and Methods section. These experiments revealed polarity impairment and consistent Rac1 behavior in both Xenopus species. The study of focal adhesion in vivo dynamics using the FAK-GFP tracer was carried out also in both species, resulting in the same phenotype. It is worth noting that the only experiment conducted exclusively in X. tropicalis was the focal adhesion disassembly assay with nocodazole.

    Regarding the improvements of the Materials and Method section see page 24, paragraph 1.

    We want to highlight that at the beginning of the Materials and Methods section, we incorporated a paragraph to clarify that "All experiments were conducted in both Xenopus species (X.t and X.l) using distinct concentrations of the morpholino (MO) and mRNA, as specified in each respective methodology description". This approach consistently yielded similar results. It is important to note that for the figures, we selected the most representative images.

    We have also specified in each figure legend which Xenopus species is depicted.

    Minor comments:

    While prior studies are referenced appropriately, and the text and figures are mostly clear and accurately presented, the following are a few suggestions that would help the authors improve the presentation of their data and conclusions:

    The cell biological experiments convincingly demonstrate that knockdown of Gai2 causes cells to move more slowly. It would be a nice addition to bring the explant experimental data back to the embryo by showing whether the slower moving NC cells in morphants eventually populate the BA. DO they cease to migrate or are they just slower getting to their destination? This could be done by performing snail2 ISH at a later stage (34-35?).

    R: We appreciate the reviewer's insightful point, and in response, we conducted the in situ hybridization assay at stages 32-36 to address this question. The result has been included in Figure S1F-H, revealing a delayed migration of cranial neural crest cells. Consequently, we have updated the text in the results section, page 6, paragraph 1, line 18:

    "In later developmental stages, such as stage 32, WISH revealed alterations in migration as well, albeit to a lesser extent compared to the early stages (22-23). This suggests a phenotype characterized by delayed migration (supplementary material Fig S1F-H)."

    There are places in the manuscript where the authors use the terms "silencing" or "suppression" of Gai2, when they really mean reduced translation - their system is not a genetic knockout, as clearly demonstrated in Figure 1H-I. I suggest that more accurate wording be used.

    R: We appreciate the reviewer's comment, and we agree that the Gαi2 morpholino impedes Gαi2 translation, leading to a reduction in Gαi2 protein expression. Consequently, we have revised the entire manuscript, replacing the terms "silencing" and "suppression" with "knockdown".

    In Figures 1-5 there are scale of bars on the cell images, but these are not defined in any of the figure legends.

    R: We value the reviewer's comment, and we have revised all the figure legends by including the scale information. Each image has been scaled to 10 µm with varying magnifications.

    The abstract is the weakest section of the manuscript, and would have greater impact if it were more clearly written.

    R: We appreciate the reviewer's comment on the abstract, and we have revised and edited it to enhance its quality.

    Abstract:

    "Cell migration is a complex and essential process in various biological contexts, from embryonic development to tissue repair and cancer metastasis. Central to this process are the actin and tubulin cytoskeletons, which control cell morphology, polarity, focal adhesion dynamics, and overall motility in response to diverse chemical and mechanical cues. Despite the well- established involvement of heterotrimeric G proteins in cell migration, the precise underlying mechanism remains elusive, particularly in the context of development. This study explores the involvement of Gαi2, a subunit of heterotrimeric G proteins, in cranial neural crest cell migration, a critical event in embryonic development. Our research uncovers the intricate mechanisms underlying Gαi2 influence, revealing a direct interaction with the microtubule-associated protein EB1, and through this with tubulin, suggesting a regulatory function in microtubule dynamics modulation. Here, we show that Gαi2 knockdown leads to microtubule stabilization, alterations in cell polarity and morphology with an increased Rac1-GTP concentration at the leading edge and cell-cell contacts, impaired cortical actin localization and focal adhesion disassembly. Interestingly, in Gai2 depleted cells RhoA-GTP was found to be reduced at cell-cell contacts and concentrated at the leading edge, providing evidence of Gαi2 significant role in polarity. Remarkably, treatment with nocodazole, a microtubule-depolymerizing agent, effectively reduces Rac1 activity, restoring cranial NC cell morphology, actin distribution, and overall migration. Collectively, our findings shed light on the intricate molecular mechanisms underlying cranial neural crest cell migration and highlight the pivotal role of Gαi2 in orchestrating microtubule dynamics through EB1 and EB3 interaction, modulating Rac1 activity during this crucial developmental process."

    Reviewer #1 (Significance (Required)):

    The molecular regulation of cell movement is a key feature of a number of developmental and homeostatic processes. While many of the proteins involved have been identified, how they interact to provide motility has not been elucidated in any great detail, particularly in embryo-derived cells (as opposed to cell lines). The results obtained from the presented experiments are novel, in-depth and provide a novel paradigm for how G proteins regulate microtubule dynamics which in turn regulate other components of the cytoskeleton required for cell movement. The results will be applicable to many migrating cell types, not just neural crest cells.

    Because of the application of the data to many types of cells that migrate, the audience is expected to include a broad array of developmental biologists, basic cell biologists and those interested in clinically relevant aberrant cell migrations.

    R: We really appreciate the reviewer positive comments and consideration

    FROM REVIEWER #2

    Reviewer: Major comments:

    The authors aim to address two issues in this manuscript: a) the role of Gai2 in neural crest development; and b) the mechanism of Gai2 function. While they have done a good job demonstrating a role of Gai2 in NC migration both in vivo and in vitro as well as the effects of Gai2 knockdown on cytoskeleton dynamics, protein distribution of selected polarity and focal adhesion molecules, and Rac1 activation, the link between Gai2 and the downstream effectors is largely correlative. Because of this, the model suggesting the sequential events flowing from Gai2 to microtubule to Rac1 to focal adhesion/actin should be modified to allow room for direct and indirect regulation at potentially multiple entry points.

    R: We appreciate the valuable comments provided by the reviewer. To further elucidate the mechanism underlying Gαi2 regulation of cranial neural crest cell migration, we have incorporated new data from interaction analysis conducted by PLA (proximity ligand assay). This analysis supports our proposed model, indicating Gαi2 interacts with EB proteins to form a complex with tubulin, thereby regulating microtubules dynamics and subsequently influencing Rac1 and RhoA activity, cell morphology (actin cytoskeleton) and cell-matrix adhesion, ultimately affecting migration. However, we cannot exclude that this regulation may also involve other intermediary proteins, such as GEFs, GAPs, GDIs, and others. Finally, as a result, we have revised our model and its description to provide a more detailed explanation of the potential mechanism in line with the reviewer suggestion. Specifically, we have edited the discussion/conclusion, model and the legend for Figure 6. Please refer to page 16 (paragraph 1, 2 and 3), 22 (paragraph 1), 23 (paragraph 1), 44 (Legend Fig. 6).

    __*Reviewer: *__Specific major comments are as the following:

    Strengths:

    -Determination of a role of Gai2 in neural crest migration is novel.

    -The effect of Gai2 knockdown on membrane protrusion morphology and microtubule stability and dynamics are demonstrated nicely.

    -Quantification of experimental perimeters has been performed throughout the manuscript in all the figures, and statistical analysis is included in the figures.

    R: We appreciate the reviewer positive comments

    Weaknesses: -The heavy focus of the study on microtubule is due to the previous publication on the function of Gai2 in regulation of microtubule during asymmetrical cell division. However, the activity of Gai2 is likely cell type-specific, as it has not been shown to control microtubule during cytokinesis in general. It is equally likely that Gai2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. The tone of the discussion should therefore be softened.

    R: We greatly appreciate and agree with the comment from the reviewer, highlighting the possibility that Gαi2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. In this regard, we have revised our manuscript to include a discussion of this point. We added the next paragraph in the Discussion/Conclusion section, page 22-23.

    "It is well established that the activity from the Rho family of small GTPases is controlling cytoskeletal organization during migration (Ridley et al., 2015). Contrariwise, it has been described in many cell types, that microtubules dynamic polymerization plays a crucial role in establishing the structural foundation for cell polarization, consequently influencing the direction of cell motility (Watanabe et al., 2005). Our results appear to align with this latter view. While it is reasonable to postulate the possibility that Gαi2 regulates Rac1 activity, subsequently influencing actin and microtubule dynamics, our findings in the context of cranial NC cells, lend support to an alternative sequence of events. Initially, Gαi2 knockdown leads to a decrease in microtubule dynamics, which in turn increase Rac-GTP towards the leading edge. This shift is accompanied by reduced levels of cortical actin and impaired focal adhesion disassembly, culminating in compromised cell migration. Notably, nocodazole, a microtubule-depolymerizing agent, not only diminishes Rac-GTP localization at the leading edge but also rescues cell morphology, restores normal cortical actin localization, and promotes focal adhesion disassembly, thereby facilitating cell migration. If Rac1 activity were indeed upstream of microtubules, it would be expected that nocodazole would not reduce Rac-GTP levels at the cell leading edge. These results suggest that the regulation of Rac1 activity may follow, rather than precede, alterations in microtubule dynamics, in the context of NC cells. Furthermore, in support of our model, our protein interaction analysis demonstrates Gαi2 interacting with microtubule components such as EB proteins and tubulin. As we already mention above, earlier studies have reported that microtubule dynamics promote Rac1 signaling at the leading edge and by releasing RhoGEFs promote RhoA signaling as well (Best et al., 1996; Garcin and Straube, 2019; Moore et al., 2013; Waterman-Storer et al., 1999). In addition, it is well-documented that RhoGEFs interact with microtubules, including bPix, a GEF for Rac1 and Cdc42, which, in turn, promotes tubulin acetylation (Kwon et al., 2020). Interestingly, in ovarian cancer cells, Gαi2 has been shown to activate Rac1 through an interaction with bPix, thereby jointly regulating migration in response to LPA (Ward et al., 2015). Taken together, these findings further support our proposed model (refer to Fig. 6)."

    The effect of rescue of NC migration with Rac1 inhibitor is marginal and the result is hard to interpret considering the inhibitor also blocks control NC migration. Either lower doses of Rac1 inhibitor can be used or the experiment can be removed from the manuscript, as Rac1 is required for membrane protrusions and the inhibitor doses can be hard to titrate.

    R: We appreciate and agree with the reviewer's comments. To address this concern and enhance clarity, we have incorporated the following paragraph into the manuscript within the Discussion section. Additionally, we have included information on the range of NSC23766 concentrations used for this analysis in the Materials and Methods section. Page 25, Explants and microdissection.

    In the results section see page 11 and 12, paragraph 2.

    "It is worth noting that we conducted Rac inhibitor NSC23766 trials at concentrations ranging from 20 nM to 50 nM for X. laevis and between 10 nM to 30 nM for X. tropicalis. In both cases, higher concentrations of the Rac inhibitor proved to be lethal (data not shown), underscoring the essential role of Rac1 in both cell migration and cell survival. Remarkably, our results show partial restoration in cranial NC cells dispersion following a 5-minute treatment with a low concentration of the Rac1 inhibitor (20 nM of NSC23766 X. laevis and 10 nM for X. tropicalis) (Fig. 3L-P, supplementary material movie S5). This suggests that these concentrations are sufficient to demonstrate that the increase in Rac1-GTP resulting from Gαi2 morpholino knockdown impairs cell migration."

    The partial rescue can be attributed to the crucial role of microtubule dynamics in cell migration, which acts upstream of Rac activity. Additionally, Rac is pivotal for the modulation of cell polarity at the leading edge of migration. It is worth emphasizing that Rac1 levels are critical for cell migration, as demonstrated by other researchers. Lower concentrations of Rac1-GTP have been shown to hinder cell migration in cells deficient in Rac1, leading to a significant reduction in wound closure and random cell migration (Steffen et al., 2013).

    "Therefore, we believe that the lower concentration of NSC23766 used in our assay was adequate to reduce the abnormal Rac1-GTP activity in the morphant NC cells. However, it is important to note that for normal NC cell, this level of reduction in Rac1-GTP activity is critical and sufficient to impair normal migration".

    See page 11 and 12, paragraph 2.

    Steffen A, Ladwein M, Dimchev GA, Hein A, Schwenkmezger L, Arens S, Ladwein KI, Margit Holleboom J, Schur F, Victor Small J, Schwarz J, Gerhard R, Faix J, Stradal TE, Brakebusch C, Rottner K. Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation. J Cell Sci. 2013 Oct 15;126(Pt 20):4572-88. doi: 10.1242/jcs.118232. Epub 2013 Jul 31. PMID: 23902686; PMCID: PMC3817791.

    Since the defects seem to result partially from the inability of the NC cells to retract and move away, it may help to either include some data on Rho activation patterns in knockdown cells or simply add some discussion about the issue.

    R: We acknowledge and sincerely appreciate the reviewer's valuable comments on this pivotal aspect, which significantly enhances our capacity to elucidate the impact of Gαi2 knockdown on cell polarity. To address this crucial point, we have introduced an experiment that examines RhoA-GTP localization under Gαi2 knockdown conditions, and we have incorporated a supplementary figure S3 into our manuscript. This newly added figure clearly demonstrates that, under Gαi2 knockdown conditions, and in contrast to control cells, RhoA-GTP localization is substantially disrupted at cell-cell contacts and now detected at the leading edge of the cell, providing compelling evidence of cell polarity defects (refer to Figure S3A-C). In response to these results, we have included a description of these findings in the Results section (please see page 10) and a dedicated paragraph in the Discussion section (please see page 19, paragraph 2, last line, page 19-21).

    Results section 1 (page 10, paragraph 1 line 6-12): "To achieve this, we explored whether Gαi2 regulates the subcellular distribution of active Rac1 and RhoA in cranial NC explants under Gαi2 loss-of-function conditions, considering their pivotal roles in cranial NC migration and contact inhibition of locomotion (CIL) (Carmona-Fontaine et al., 2011; Moore et al., 2013; Leal et al., 2018). Hence, we employed mRNA encoding the small GTPase-based probe, enabling specific visualization of the GTP-bound states of these proteins."

    Results section 2 (page 10, paragraph 1 line 14-27): "Consistent with earlier observations by Carmona-Fontaine et al. (2011), in control cranial NC cells, active Rac1 displayed prominent localization at the leading edge of migrating cells, whereas its presence was reduced at cell-cell contacts, coincident with an increase in RhoA-GTP levels (white arrows in Fig. 3A, supplementary material Figure S3A,C). On the contrary, in comparison to the control cells, Gαi2 morphants exhibit a pronounced accumulation of active Rac1 protein in the protrusions at cell-cell contacts, where active RhoA localization is conventionally expected (white arrow in Fig. 4B, supplementary material Figure S3A,C and movie S4). In contrast to control cells, a notable shift in the localization of active RhoA protein was observed, with its predominant accumulation now detected at the leading edge of the cell, instead of the typical localization towards the trailing edge or cell-cell contacts (__supplementary material Figure S3B,C). __These findings suggest a dysregulation of contractile forces that align with the observed distribution of active RhoA, cortical actin disruption, and diminished retraction in cell treated with Gαi2MO."

    *Discussion section: (page 19 last line, page 20, paragraph 1, line 1-20) *

    "Other studies have reported that microtubule assembly promotes Rac1 signaling at the leading edge, while microtubule depolymerization stimulates RhoA signaling through guanine nucleotide exchange factors associated with microtubule-binding proteins controlling cell contractility, via Rho-ROCK and focal adhesion formation (Krendel et al., 2002; Ren et al., 1999; Best et al., 1996; Garcin and Straube, 2019; Waterman-Storer et al., 1999; Bershadsky et al., 1996; Moore et al., 2013). This mechanism would contribute to establishing the antero-posterior polarity of cells, crucial for maintaining migration directionality, underscoring the significance of regulating microtubule dynamics in directed cell migration. These findings closely align with the results obtained in this investigation, demonstrating that Gαi2 loss of function reduces microtubule catastrophes and promotes tubulin stabilization, resulting in increased localization of active Rac1 at the leading edge and cell-cell contacts, while decreasing active RhoA at the cell-cell contact but increasing it at the leading edge. This possibly reinforces focal adhesion, which is consistent with the presence of large and highly stable focal adhesions under Gαi2 knockdown conditions. This finding also suggests a dysregulation of contractile forces in comparison to control cells, a result that aligns with the observed distribution of active RhoA, cortical actin distribution and diminished retraction in cells treated with Gαi2MO. This strikingly contrasts with the normal cranial NC migration phenotype, where Rac1 is suppressed while active RhoA is increased at cell-cell contacts during CIL, leading to a shift in polarity towards the cell-free edge to sustain directed migration (Theveneau et al., 2010; Shoval and Kalcheim, 2012; Leal et al., 2018)."

    To consider focal adhesion dynamics, live imaging should be used in the analysis. The fixed samples are different from each other, and natural variations of focal adhesion may exist among the samples. This can obscure data collection and quantification.

    R: We agree with the reviewer that focal adhesion (FA) dynamics need to be analysed using live imaging. Indeed, Fig 5E-H shows an extensive analysis of FA using live imaging of neural crest expressing FAK-GFP. As complement to this live imaging analysis, and in order to analyse the effect on the endogenous levels of FA proteins, we performed immunostaining against FA. Both experiments using live imaging or fixed cells produce similar results, and they are consistent with our model on the role of Gαi2 on FA dynamics.

    Reviewer: minor comments

    Fig. 2, the centrosomes in control cells are not always obvious. The microtubules simply seem to be more networked and more fluid in control cells. This should be clarified with either marking the centrosomes in the figure or modifying the wording in the manuscript.

    R: We appreciate and concur with the reviewer's comment on this matter. As pointed out by the reviewer, the precise localization of the centrosome is not consistently clear in all cells. In response to this observation, we have revised the manuscript to emphasize this aspect solely as "microtubule morphology". Please refer to the Results section description Figure 2.

    In Fig. 3, a better negative control for co-IP should be using anti-V5 antibody to IP against tubulin/EB1/EB3 in the absence of Gai2-V5.

    R: We appreciate the reviewer's comment, and we agree with the suggested control. Accordingly, we have included this control in Supplementary material Figure S4A. Additionally, we conducted all Co-IPP in triplicate, and these data have been incorporated into Supplementary material Figure S4B. Furthermore, as mentioned earlier, we have reorganized some of the sections of the results to improve the logical flow of the manuscript's description. As a result, the Figure presenting the interaction analysis by Co-IPP now corresponds to Figure 5.

    The data for cell polarity proteins Par3 and PKC-zeta seem to be out of place. It is unclear whether mis-localization of these proteins has anything to do with NC migration defects induced by Gai2 knockdown. The conclusion does not seem to be affected if the data are taken out of the manuscript.

    R: We appreciate the reviewer's concern, and we would like to highlight two points in this regard. Firstly, we have included these results as additional data to support the impact of Gai2 knockdown on cell polarity, given that these two proteins are commonly used as polarity markers. Secondly, we have discussed this aspect extensively in the Discussion section of the manuscript. (See page 20, paragraph 1, lines 21-31).

    In that section, we delve into the relationship between aPKC, Par3, and Gαi2 in controlling cell polarity during asymmetric cell division, as described in Hao et al., 2010. Par3 is known to play a role in regulating microtubule dynamics and Rac1 activation through its interaction with Rac-GEF Tiam1 (Chen et al., 2005). Additionally, it has been shown to promote microtubule catastrophes and inhibit Rac1/Trio signaling, regulating Contact Inhibition of Locomotion (CIL) as demonstrated in Moore et al., 2013. Thus, we believe that the data we present support the relationship between Par3 and aPKC localization changes and the neural crest migration defects induced by Gαi2 knockdown, probably by controlling microtubule dynamics. However, we have moved these results as part of the supplementary Figure S3D-G.

    In Suppl. Fig. 1, protrusion versus retraction should be defined more clearly. The retraction shown in this figure seems to be just membrane between protrusions instead of actively retracting membrane.

    R: We appreciate the reviewer's comments, and here we aim to provide a clearer description of our approach to this analysis. For the measurement of protrusion extension/retraction, we conducted two distinct experiments. The first, as described in Figure 1, involved measuring membrane extension and retraction in live cell using membrane-GFP by utilizing the image subtraction tool in ImageJ, which highlights changes in the membrane in red. Secondly, we employed ADAPT software to quantify cell perimeter based on fluorescence intensity in live cell using lifeactin-GFP, distinguishing membrane extension in green and retraction in red (as has been shown similarly in Barry et al., 2015). In both approaches, we observed a substantial increase in membrane protrusion (both in area and extension) and protrusion stability in Gαi2 morphants. Hence, we have revised the Materials and Methods section of the manuscript and included this clarification.

    See Materials and Methods section, Cell dispersion and morphology, page 28.

    In addition we inform hat this images now are included in Supplementary material Fig S2G,H.

    Barry DJ, Durkin CH, Abella JV, Way M. Open source software for quantification of cell migration, protrusions and fluorescence intensities. J Cell Biol. 2015. Doi: 10.1083/jcb.201501081

    Discussion can be improved by better incorporating all the components to make a cohesive story on how Gai2 works to regulate migration in the context of the neural crest cells.

    R: We appreciate the reviewer's comment and agree. To enhance the manuscript, we have included a new paragraph at the end of the Discussion/Conclusion section specifically addressing this point. For more details, please refer to page 23.

    "Therefore, in the context of collective cranial NC cells migration, our findings reveal the pivotal role played by Gαi2 in orchestrating the intricate interplay of microtubule dynamics and cellular polarity. When Gαi2 levels are diminished, we observe significant impediments in the ability of cells to efficiently navigate through their environment, resulting in a range of distinct effects. First and foremost, Gαi2 deficiency leads to the diminished ability of cells to adjust and reorient new protrusions effectively. Primary protrusions exhibit higher stability and heightened levels of active Rac1/RhoA when compared to control conditions in the leading edge. In addition, we observe a notable increase in protrusion area, a decrease in retraction velocity, and an enhanced level of cell-matrix adhesion in Gαi2 knockdown cells. These findings underscore the pivotal role that Gαi2 plays in the modulation of various cellular dynamics essential for collective cranial NC cells migration. Notably, the application of nocodazole, a microtubule-depolymerizing agent, and NSC73266, a Rac1 inhibitor, to Gαi2 knockdown cells leads to the rescue of the observed effects, thus facilitating migration. This observed response closely mirrors the outcomes associated with Par3, a known regulator of microtubule catastrophe during contact inhibition of locomotion (CIL) in NC cells (Moore et al., 2013). This parallel implies that there exists a delicate equilibrium between microtubule dynamics and Rac1-GTP levels, crucial for the establishment of proper cell polarity during collective migration. Our findings collectively position Gαi2 as a central master regulator within the intricate framework of collective cranial NC migration. This master regulator role is pivotal in orchestrating the dynamics of polarity, morphology, and cell-matrix adhesion by modulating microtubule dynamics through interactions with EB1 and EB3 proteins, described here for the first time, possible in a protein complex involving other intermediary proteins such as other microtubules accessory proteins like CLIP170, actin intermediaries, like mDia1-2, and signaling proteins such as GDIs, GAPs and GEFs, thus fostering crosstalk between the actin and tubulin cytoskeletons. This orchestration ultimately ensures the effective collective migration of cranial NC cells (Fig. 6)."

    Review____er #2 (Significance (Required)):

    The authors demonstrate a role of Gai2 in regulation of neural crest migration in Xenopus by modulating microtubule dynamics. In addition, they show an effect of Gai2 knockdown on Rac1 spatial activation and focal adhesion stability. These are novel discoveries of the study. Some limitations exist in linking Gai2 with downstream effectors that directly or indirectly impact on cytoskeleton and Rac1 small GTPase.

    R: We really appreciate the reviewer positive comments and consideration. We believe that the review process has significantly strengthened our manuscript in this regard.

    FROM REVIEWER #3

    __ ____Reviewer: mayor comments:__

    The authors focus exclusively on the analysis of the subcellular levels of Rac1, which is probably related to the fact that they observe large extended protrusions with high Rac1 activity. However, as the authors note, a global fine-tuning of Rho GTPase activity is required for neural crest migration. One of the observed phenotypes of Gαi2-morphant neural crest cells is a decrease in cell dispersion, which may be caused by defects in contact inhibition of locomotion (CIL). This process involves a local activation of RhoA at cell-cell contact sites (Carmona-Fontaine et al., 2008). Furthermore, in fibroblast, RhoA/ROCK activity is required for the front-rear polarity switch during CIL (Kadir et al., 2011). Interestingly, similar to the Gαi2 loss of function phenotype, ROCK inhibition leads to microtubule stabilization, which can be rescued by nocodazole treatment, restoring microtubule dynamics and CIL. Therefore, it would also be interesting to know how RhoA activity is affected in Gαi2-morphant NC cells. At a minimum, this point should be be included in the discussion.

    R: We acknowledge and sincerely appreciate the reviewer's valuable comments on this pivotal aspect, which significantly enhances our capacity to elucidate the impact of Gαi2 knockdown on cell polarity. To address this crucial point, we have introduced an experiment that examines RhoA-GTP localization under Gαi2 knockdown conditions, and we have incorporated a supplementary figure S3A-C into our manuscript. This newly added figure clearly demonstrates that, under Gαi2 knockdown conditions and in contrast to control cells, RhoA-GTP localization is substantially disrupted at cell-cell contacts and now detected at the leading edge of the cell, providing compelling evidence of cell polarity defects (refer to Figure S3). In response to these results, we have included a description of these findings in the Results section (please see page 10) and a dedicated paragraph in the Discussion section (please see page 19-20).

    Results section 1 (page 10, paragraph 1 line 6-12): "To achieve this, we explored whether Gαi2 regulates the subcellular distribution of active Rac1 and RhoA in cranial NC explants under Gαi2 loss-of-function conditions, considering their pivotal roles in cranial NC migration and contact inhibition of locomotion (CIL) (Carmona-Fontaine et al., 2011; Moore et al., 2013; Leal et al., 2018). Hence, we employed mRNA encoding the small GTPase-based probe, enabling specific visualization of the GTP-bound states of these proteins."

    Results section 2 (page 10, paragraph 1 line 14-27): "Consistent with earlier observations by Carmona-Fontaine et al. (2011), in control cranial NC cells, active Rac1 displayed prominent localization at the leading edge of migrating cells, whereas its presence was reduced at cell-cell contacts, coincident with an increase in RhoA-GTP levels (white arrows in Fig. 3A, supplementary material Figure S3A,C). On the contrary, in comparison to the control cells, Gαi2 morphants exhibit a pronounced accumulation of active Rac1 protein in the protrusions at cell-cell contacts, where active RhoA localization is conventionally expected (white arrow in Fig. 4B, supplementary material Figure S3A,C and movie S4). In contrast to control cells, a notable shift in the localization of active RhoA protein was observed, with its predominant accumulation now detected at the leading edge of the cell, instead of the typical localization towards the trailing edge or cell-cell contacts (__supplementary material Figure S3B,C). __These findings suggest a dysregulation of contractile forces that align with the observed distribution of active RhoA, cortical actin disruption, and diminished retraction in cell treated with Gαi2MO."

    *Discussion section: (page 19, second paragraph, line 12 and page 20, paragraph 1, line 1-18) *

    "Other studies have reported that microtubule assembly promotes Rac1 signaling at the leading edge, while microtubule depolymerization stimulates RhoA signaling through guanine nucleotide exchange factors associated with microtubule-binding proteins controlling cell contractility, via Rho-ROCK and focal adhesion formation (Krendel et al., 2002; Ren et al., 1999; Best et al., 1996; Garcin and Straube, 2019; Waterman-Storer et al., 1999; Bershadsky et al., 1996; Moore et al., 2013). This mechanism would contribute to establishing the antero-posterior polarity of cells, crucial for maintaining migration directionality, underscoring the significance of regulating microtubule dynamics in directed cell migration. These findings closely align with the results obtained in this investigation, demonstrating that Gαi2 loss of function reduces microtubule catastrophes and promotes tubulin stabilization, resulting in increased localization of active Rac1 at the leading edge and cell-cell contacts, while decreasing active RhoA at the cell-cell contact but increasing it at the leading edge. This possibly reinforces focal adhesion, which is consistent with the presence of large and highly stable focal adhesions under Gαi2 knockdown conditions. This finding also suggests a dysregulation of contractile forces in comparison to control cells, a result that aligns with the observed distribution of active RhoA, cortical actin distribution and diminished retraction in cells treated with Gαi2MO. This strikingly contrasts with the normal cranial NC migration phenotype, where Rac1 is suppressed while active RhoA is increased at cell-cell contacts during CIL, leading to a shift in polarity towards the cell-free edge to sustain directed migration (Theveneau et al., 2010; Shoval and Kalcheim, 2012; Leal et al., 2018)."

    The co-Immunoprecipitation data lack marker bands (larger images/sections of the blots would be preferable) and the labelling is not clear. What do the white arrows in Fig. 3H,I mean? What does "elu" and "non eluted" mean?. ? Did the reverse IP work as well?

    R: We appreciate the reviewer's comments, and here we intend to provide a more detailed explanation of our approach to this analysis. Since we do not possess a secondary antibody specific to the heavy chain, our method involves eluting the co-immunoprecipitated proteins to visualize those with weights close to that of the light chain (such as EB1). We have outlined this elution step in the "Cell lysates and co-immunoprecipitation" protocol in the Materials and Methods section. To ensure proper control, we load both fractions - the eluted (or supernatant) and non-eluted (or resin) fractions - to monitor the amount of protein extracted from the resin using a 1% SDS solution. It's important to note that the elution step, as indicated by the V5 signal, is not entirely efficient, and a significant portion of the protein remains bound to the resin. This issue may also apply to the EB1 protein; however, it is still possible to visualize both bands (Gαi2V5 and EB1).

    As we mentioned earlier the Co-IPP analysis now are in Figure 5. We have revised the legend for Figure 5 to include an explanation of the terms 'elu' (eluted fraction) and 'non-eluted' (non-eluted fraction). We have also included the explanation of the white arrows' significance in the legends for Figure 5H and 5I. These arrows indicate the bands corresponding to the immunoprecipitated proteins.

    We also agree with the reviewer's suggestion to conduct the reverse IP. To address this point, and in favour of the lack of this control, accordingly, we have included a negative control for co-IP using anti-V5 antibody to IP, this control was included in Supplementary material Figure S4A. Additionally, we conducted all Co-IPP in triplicate, and these data have been incorporated into Supplementary material Figure S4B.

    The presentation of the Delaunay triangulations varies in quality. In Fig. 1 J/K the cells are clearly visible in the images, while this is not the case in Fig. 3 J-M and Fig. 4K-N. Conversely, the Delaunay triangulations in Fig. 1L are mainly black, while they are clear in Fig. 3 and 4. Perhaps the authors could find a more consistent way to present the data. Were the explants all approximately the same size at the beginning of the experiment? The Gαi2-morphant explant in Fig. 3K appears to be unusually small.

    R: We appreciate the reviewer's concerns and have taken steps to address them. To improve the quality of our data, we have made enhancements to the presentation of Figures 3 (panels L-O) and Figure 5 (panels P-S). Specifically, we have standardized the Delaunay triangulation representations.

    Regarding the size of the explants at the beginning of the experiments, they were indeed approximately similar in size. We confirmed this by including a reference point (point 0) for each condition in the figures 5. However, in the panels presented, we show the results after 10 hours (Figure 5, X. laevis, in the actual Figure organization) and 4 hours (Figure 3, X. tropicalis, in the actual Figure organization) to assess cell dispersion, as indicated in the respective figure legends. This uniformity in size was further ensured by the calculation used to quantify dispersion. For the dispersion assay, we normalized each initial size of the explant upon the control, and we have added another representative explant of Gαi2 morpholino with its Delaunay triangulation to facilitate the experiment interpretation. Every Delaunay triangulation calculates the area generated between three adjacent cells and it grows depending on how much disperse are the cells between each other in the final point. (See Material and Methods section, Cell dispersion and morphology). As we can see in the manuscript, in every dispersion experiment that we have performed with Gαi2 morpholino, the cells cannot disperse at all. Furthermore, to analyze the dispersion rate in this experiment we use Control n= 21 explants, Gαi2MO n= 24 explants, Gαi2MO + 65 nM Nocodazole n= 36 explants, Control + 65 nM Nocodazole n= 30 explants (as we mentioned in the manuscript legend).

    Why was the tubulin distribution in Fig. 2F measured from the nucleus to the cell cortex? Would it not make more sense to include cell protrusions? This does not seem to be the case in the example shown in Fig. 2F.

    R: We appreciate the reviewer's concern. We would like to clarify that for the tubulin distribution measurements, we indeed measured from the nucleus to the cell protrusion. As a result, we have made an edit to Figure 2 (panel 2F) to provide further clarity on this matter.

    The immunostaining for acetylated tubulin (Fig. 3A,B) looks potentially unspecific and seems to co-localize with actin (for comparison see Bance et al., 2019). For imaging and quantification, it may be better to use tubulin co-staining to calculate the percentage of acetylated tubulin. Please also add marker bands to the Western blot in Fig. 3C. If this issue cannot be resolved it may be better to only include the Western blot data.

    R: We appreciate the reviewer's concern about the potential unspecific nature of acetylated-tubulin and its co-localization with actin. Regarding the co-localization with actin, it is predominantly observed in panel B, and we attribute this phenomenon to the Gαi2 morphant phenotype, where cortical actin is notably reduced, creating the appearance of co-localization. In response to the reviewer comment, we have retained the acetylated tubulin western blot analysis in the main Figure 5A,B, while relocating the immunofluorescence analysis to Supplementary material Figure S4C-H. Additionally, we have included the measurements of the acetylated tubulin fluorescence intensity for both conditions Gαi2MO and control, as depicted Supplementary material Figure S4I.

    We have also included marker weight indications on the western blot panel in now Figure 5A.

    The model in Fig.6 indicates that Gαi2 inhibits EB1/3. What is the experimental evidence and the proposed mechanism for this? In the discussion, the authors cite evidence that Gαi activates the intrinsic GTPase activity of tubulin, which would destabilize microtubules by removing the GTP cap. However, this mechanism would not directly affect EB1 and EB3 stability as the Fig. 6A seems to suggest. The authors also mention that EB3 appears to be permanently associated with microtubules in Gαi2-morphant cells. How would this work, given that end-binding proteins bind to the cap region? Are the authors suggesting that there is an extended cap region in Gαi2 morphants?

    R: We appreciate the reviewer's valuable comments. We have revised our model accordingly to our data and new data that we have incorporated regarding interaction analysis conducted by PLA (proximity ligand assay), in order to further elucidate the mechanism underlying Gαi2 regulation of cranial neural crest cell migration. This analysis supports our actual proposed model, indicating Gαi2 interacts with EB proteins to form a complex with tubulin, thereby regulating microtubules dynamics and subsequently influencing Rac1 and RhoA activity, cell morphology (actin cytoskeleton) and cell-matrix adhesion, ultimately affecting migration. Therefore, we have revised our model and its description to provide a more detailed explanation of the potential mechanism in line with the reviewer suggestion. Specifically, we have edited the discussion/conclusion, model and the legend for Figure 6. Please refer to page 16 (paragraph 1, 2 and 3), 22 (paragraph 1), 23 (paragraph 1), 45 (Legend Fig. 6). In addition, in Gαi2 knockdown conditions we have found a strong reduction in microtubules dynamics following EB3-GFP comets. Regarding the observation that EB3 seems to be persistently associated with microtubules in Gαi2-morphant cells, we wish to clarify that this is a speculation based on the microtubule phenotype observed during our dynamic analysis, where they appear more like lines rather than comets. It is important to note that none of the experiments conducted in this study conclusively demonstrate this, and thus, it remains a suggestion. As a result, we have revised our model in accordance with the reviewer suggestion.

    Reviewer 3: minor comments:

    The citation of Wang et al. 2018 in the introduction does not seem to fit.

    R: We appreciate the correction provided by the reviewer. We have carefully reviewed the Introduction and Reference sections and have corrected this error.

    Does the graph in Fig. 4S show average values for the three conditions? If so, what is the standard deviation?

    R: We appreciate the reviewer's concern and we have added the standard deviation to now Figure 4J.

    From the images in Fig. 2G and H, it is difficult to understand what the difference is between the four groups shown.

    R: We appreciate the reviewer's comment, and to clarify this point, we would like to explain that the comparison has been made between each type of comet. The PlusTipTracker software separates comets based on their speed and lifetime, classifying them as fast long-lived, fast short-lived, slow long-lived, or slow short-lived. In both conditions (control and morphant cells), we compared the percentage of each type of comet, as previously described in Moore et al., 2013. The results demonstrate that morphant cells exhibit an increase in slow comets compared to control cells. The same explanation is described in the Material and Methods section on page 28, Microtubule dynamics analysis.

    Review____er #3: (Significance (Required)):

    Overall, the study is well executed and significantly advances our understanding of the control and role of microtubule dynamics in cell migration, which is much less understood compared to the function of the actin cytoskeleton in this process. The strength of the study is the use of state-of-the-art (live imaging) techniques to characterize the role of Gαi in neural crest migration at the cellular/subcellular level. This article will be of interest to a broad readership, including researchers interested in basic embryonic morphogenesis, cell migration, and cytoskeletal dynamics, as well as translational/clinical researchers interested in cancer progression or wound healing.

    R: We really appreciate the reviewer positive comments and consideration. We believe that the review process has significantly strengthened our manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: The manuscript by Villaseca et al. analyzes the role of Gαi2 in cranial neural crest migration and reveals a novel mechanistic link to microtubule dynamics. The authors nicely demonstrate that Gαi2 is required for Xenopus neural crest migration and affects cell dispersion, cell polarity, focal adhesion turnover, and microtubule dynamics. They find that Gαi2-morphant neural crest cells are elongated, have larger, more stable protrusions, higher active Rac1 levels, and a concentration of microtubules at the leading edge. Using co-immunoprecipitation, the authors show that Gαi2 forms a complex with α-tubulin and the microtubule plus-end binding proteins EB1 and EB3, which are known regulators of microtubule dynamics. Time-lapse imaging shows that Gαi2 loss of function increases microtubule stability, which is further supported by an increase in acetylated tubulin levels. Consistently, treatment with nocodazole, which inhibits microtubule polymerization, as well as treatment with a Rac1 inhibitor, is able to rescue cell dispersion and morphology of Gαi2-morphant neural crest cells. The authors propose a model, whereby Gαi2 interacts with components of the plus-tip microtubule-binding complex to control microtubule dynamics and Rac1 activity to establish cell polarity, disassemble focal adhesion, and thereby facilitate neural crest migration.

    Major comments:

    1. The authors focus exclusively on the analysis of the subcellular levels of Rac1, which is probably related to the fact that they observe large extended protrusions with high Rac1 activity. However, as the authors note, a global fine-tuning of Rho GTPase activity is required for neural crest migration. One of the observed phenotypes of Gαi2-morphant neural crest cells is a decrease in cell dispersion, which may be caused by defects in contact inhibition of locomotion (CIL). This process involves a local activation of RhoA at cell-cell contact sites (Carmona-Fontaine et al., 2008). Furthermore, in fibroblast, RhoA/ROCK activity is required for the front-rear polarity switch during CIL (Kadir et al., 2011). Interestingly, similar to the Gαi2 loss of function phenotype, ROCK inhibition leads to microtubule stabilization, which can be rescued by nocodazole treatment, restoring microtubule dynamics and CIL. Therefore, it would also be interesting to know how RhoA activity is affected in Gαi2-morphant NC cells. At a minimum, this point should be be included in the discussion.
    2. The co-Immunoprecipitation data lack marker bands (larger images/sections of the blots would be preferable) and the labelling is not clear. What do the white arrows in Fig. 3H,I mean? What does "elu" and "non eluted" mean? Did the reverse IP work as well?
    3. The presentation of the Delaunay triangulations varies in quality. In Fig. 1 J/K the cells are clearly visible in the images, while this is not the case in Fig. 3 J-M and Fig. 4K-N. Conversely, the Delaunay triangulations in Fig. 1L are mainly black, while they are clear in Fig. 3 and 4. Perhaps the authors could find a more consistent way to present the data. Were the explants all approximately the same size at the beginning of the experiment? The Gαi2-morphant explant in Fig. 3K appears to be unusually small.
    4. Why was the tubulin distribution in Fig. 2F measured from the nucleus to the cell cortex? Would it not make more sense to include cell protrusions? This does not seem to be the case in the example shown in Fig. 2F.
    5. The immunostaining for acetylated tubulin (Fig. 3A,B) looks potentially unspecific and seems to co-localize with actin (for comparison see Bance et al., 2019). For imaging and quantification, it may be better to use tubulin co-staining to calculate the percentage of acetylated tubulin. Please also add marker bands to the Western blot in Fig. 3C. If this issue cannot be resolved it may be better to only include the Western blot data.
    6. The model in Fig.6 indicates that Gαi2 inhibits EB1/3. What is the experimental evidence and the proposed mechanism for this? In the discussion, the authors cite evidence that Gαi activates the intrinsic GTPase activity of tubulin, which would destabilize microtubules by removing the GTP cap. However, this mechanism would not directly affect EB1 and EB3 stability as the Fig. 6A seems to suggest. The authors also mention that EB3 appears to be permanently associated with microtubules in Gαi2-morphant cells. How would this work, given that end-binding proteins bind to the cap region? Are the authors suggesting that there is an extended cap region in Gαi2 morphants?

    Minor comments

    1. The citation of Wang et al. 2018 in the introduction does not seem to fit.
    2. Does the graph in Fig. 4S show average values for the three conditions? If so, what is the standard deviation?
    3. From the images in Fig. 2G and H, it is difficult to understand what the difference is between the four groups shown.

    Referees cross-commenting The concerns raised by my colleagues are entirely valid.

    Significance

    Overall, the study is well executed and significantly advances our understanding of the control and role of microtubule dynamics in cell migration, which is much less understood compared to the function of the actin cytoskeleton in this process. The strength of the study is the use of state-of-the-art (live imaging) techniques to characterize the role of Gαi in neural crest migration at the cellular/subcellular level. This article will be of interest to a broad readership, including researchers interested in basic embryonic morphogenesis, cell migration, and cytoskeletal dynamics, as well as translational/clinical researchers interested in cancer progression or wound healing.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    The manuscript by Villaseca et al. describes functional analysis of Gai2 in cranial neural crest (CNC) migration using the frog Xenopus as their model system. The authors performed the loss-of-function assay to knock down expression of endogenous of Gai2 and discovered that CNC migration was impaired in the absence of changes of CNC fate specification. Based on the literature on Gai2 activities in other cellular contexts, the authors speculated that Gai2 might regulate microtubule dynamics and Rac1 function. Their studies using immunofluorescence (IF) and live-cell imaging indeed showed that microtubules were stabilized in membrane protrusions with concurrent activation of Rac1 in Gai2 knockdown cells. In addition, focal adhesion turnover was reduced. They further demonstrated that the CNC migration defects could be partially rescued by destabilization of microtubules with chemical treatment. The authors conclude from the studies that Gai2 orchestrates microtubule dynamics and modulates Rac1 activation during neural crest migration.

    Major comments

    The authors aim to address two issues in this manuscript: a) the role of Gai2 in neural crest development; and b) the mechanism of Gai2 function. While they have done a good job demonstrating a role of Gai2 in NC migration both in vivo and in vitro as well as the effects of Gai2 knockdown on cytoskeleton dynamics, protein distribution of selected polarity and focal adhesion molecules, and Rac1 activation, the link between Gai2 and the downstream effectors is largely correlative. Because of this, the model suggesting the sequential events flowing from Gai2 to microtubule to Rac1 to focal adhesion/actin should be modified to allow room for direct and indirect regulation at potentially multiple entry points.

    Specific major comments are as the following:

    Strengths:

    -Determination of a role of Gai2 in neural crest migration is novel. -The effect of Gai2 knockdown on membrane protrusion morphology and microtubule stability and dynamics are demonstrated nicely. -Quantification of experimental perimeters has been performed throughout the manuscript in all the figures, and statistical analysis is included in the figures.

    Weaknesses:

    • The heavy focus of the study on microtubule is due to the previous publication on the function of Gai2 in regulation of microtubule during asymmetrical cell division. However, the activity of Gai2 is likely cell type-specific, as it has not been shown to control microtubule during cytokinesis in general. It is equally likely that Gai2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. The tone of the discussion should therefore be softened.
    • The effect of rescue of NC migration with Rac1 inhibitor is marginal and the result is hard to interpret considering the inhibitor also blocks control NC migration. Either lower doses of Rac1 inhibitor can be used or the experiment can be removed from the manuscript, as Rac1 is required for membrane protrusions and the inhibitor doses can be hard to titrate.
    • Since the defects seem to result partially from the inability of the NC cells to retract and move away, it may help to either include some data on Rho activation patterns in knockdown cells or simply add some discussion about the issue.
    • To consider focal adhesion dynamics, live imaging should be used in the analysis. The fixed samples are different from each other, and natural variations of focal adhesion may exist among the samples. This can obscure data collection and quantification.

    Minor comments

    • Fig. 2, the centrosomes in control cells are not always obvious. The microtubules simply seem to be more networked and more fluid in control cells. This should be clarified with either marking the centrosomes in the figure or modifying the wording in the manuscript.
    • In Fig. 3, a better negative control for co-IP should be using anti-V5 antibody to IP against tubulin/EB1/EB3 in the absence of Gai2-V5.
    • The data for cell polarity proteins Par3 and PKC-zeta seem to be out of place. It is unclear whether mis-localization of these proteins has anything to do with NC migration defects induced by Gai2 knockdown. The conclusion does not seem to be affected if the data are taken out of the manuscript.
    • In Suppl. Fig. 1, protrusion versus retraction should be defined more clearly. The retraction shown in this figure seems to be just membrane between protrusions instead of actively retracting membrane.
    • Discussion can be improved by better incorporating all the components to make a cohesive story on how Gai2 works to regulate migration in the context of the neural crest cells.

    Referees cross-commenting I agree with other reviewers' comments.

    Significance

    The authors demonstrate a role of Gai2 in regulation of neural crest migration in Xenopus by modulating microtubule dynamics. In addition, they show an effect of Gai2 knockdown on Rac1 spatial activation and focal adhesion stability. These are novel discoveries of the study. Some limitations exist in linking Gai2 with downstream effectors that directly or indirectly impact on cytoskeleton and Rac1 small GTPase.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This manuscript examines the role of a G protein, Gai2, in regulating the migration of cranial neural crest cells. Although previous literature has established that heterotrimeric G proteins are involved in cell migration, a central process during embryogenesis and adult homeostasis, the underlying cell biological mechanisms of their activities have not been elucidated. This manuscript rigorously examines the various aspects of Gai2 protein interactions to generate an exciting new paradigm in which Gai2 maintains normal microtubule dynamics by binding to tubulin and EB proteins. This normally dynamic microtubular intracellular environment then promotes cortical actin formation in the leading edge of the migrating cell as well as rapid focal adhesion disassembly by controlling Rac1 activity. Under conditions in which the levels of Gai2 are reduced by MO-mediated knockdown, cells display reduced microtubule dynamics and a decreased catastrophe rate, resulting in slower and more stable microtubules to which EB3 is more persistently associated. A stable microtubule environment leads to enhanced Rac1 activation at the leading edge and stable and larger focal adhesions, resulting in reduced migration. The authors utilize cutting edge approaches to examine the interactions between Gai2 and these other cellular components, taking advantage of the well characterized cell migration model - the cranial neural crest - both in embryos and in cultured explants of these cells.

    Major comments:

    The manuscript is mostly well written (it could use a few minor grammatical corrections), the significance of the problem is well described, and the results are clearly presented with adequate controls. The movies, provided as supplementary material, are of the highest quality and are essential additions to the stills provided in the figures. The data convincingly support the key conclusions of the manuscript.

    Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No

    Would additional experiments be essential to support the claims of the paper? No additional experiments are essential.

    Are the experiments adequately replicated and statistical analysis adequate? The number of embryos/ explants per assay and the number of explant replicates for each assay and the statistical assessments are rigorous.

    Are the data and the methods presented in such a way that they can be reproduced? Mostly, however, the description of the MO used for Gai2 knockdown needs more detail:

    1. Does the MO knockdown both S and L homoeologs of X. laevis? Since the level of GAPDH in Figure 1H also looks reduced in Gai2 MO lane, it should be made clear that the apparent knockdown of Gai2 was normalized to GAPDH, rather than being the results of unequal loading of the gel. Yes, I recognize that Figure 1I says normalized, but this is not stated in the results or the methods. Also, was this experiment done with X. laevis or X. tropicalis? I could imagine that if done in X. laevis, the lack of complete knockdown might be due to only one homoeolog being affected.
    2. The knowledge of the efficacy of knockdown in each Xenopus species provided by the information requested in the previous point, would allow the reader to assess the level of knockdown in the remaining assays. To do this, the authors should tell us which assays were done in which species. I am not suggesting that each experiment needs to be done in each species, only that the information should be provided. If the MO is more effective in X. tropicalis - which assays used this species? If the knock down is partial, as shown in Figure 1H-I, which species this represents in the remaining assays would be useful knowledge.

    Minor comments:

    While prior studies are referenced appropriately, and the text and figures are mostly clear and accurately presented, the following are a few suggestions that would help the authors improve the presentation of their data and conclusions:

    1. The cell biological experiments convincingly demonstrate that knockdown of Gai2 causes cells to move more slowly. It would be a nice addition to bring the explant experimental data back to the embryo by showing whether the slower moving NC cells in morphants eventually populate the BA. DO they cease to migrate or are they just slower getting to their destination? This could be done by performing snail2 ISH at a later stage (34-35?)
    2. There are places in the manuscript where the authors use the terms "silencing" or "suppression" of Gai2, when they really mean reduced translation - their system is not a genetic knockout, as clearly demonstrated in Figure 1H-I. I suggest that more accurate wording be used.
    3. In Figures 1-5 there are scale of bars on the cell images, but these are not defined in any of the figure legends.
    4. The abstract is the weakest section of the manuscript, and would have greater impact if it were more clearly written.

    Referees cross-commenting

    The concerns are fair assessments. However, most can be addressed in the text and by clearer presentation of existing data rather than more experimentation.

    Significance

    The molecular regulation of cell movement is a key feature of a number of developmental and homeostatic processes. While many of the proteins involved have been identified, how they interact to provide motility has not been elucidated in any great detail, particularly in embryo-derived cells (as opposed to cell lines). The results obtained from the presented experiments are novel, in-depth and provide a novel paradigm for how G proteins regulate microtubule dynamics which in turn regulate other components of the cytoskeleton required for cell movement. The results will be applicable to many migrating cell types, not just neural crest cells.

    Because of the application of the data to many types of cells that migrate, the audience is expected to include a broad array of developmental biologists, basic cell biologists and those interested in clinically relevant aberrant cell migrations.

    Reviewer keywords: Xenopus embryology; neural crest gene expression; use of MO-mediated knockdown of gene expression. Not an expert in microtubule dynamics.

  5. Version published to 10.1101/2023.09.07.556733v3 on bioRxiv
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    Reply to the reviewers

    Response to reviewer comments

    R: We really appreciate the reviewer positive comments and consideration, and we believe that the review process has significantly strengthened our manuscript.

    We have responded to all the reviewer comments, as follows:

    Response (R)

    From Reviewer #1

    Major comments: The manuscript is mostly well written (it could use a few minor grammatical corrections), the significance of the problem is well described, and the results are clearly presented with adequate controls. The movies, provided as supplementary material, are of the highest quality and are essential additions to the stills provided in the figures. The data convincingly support the key conclusions of the manuscript.

    R: We really appreciate the reviewer positive comments, and we have revised the manuscript accordingly

    1. Does the MO knockdown both S and L homoeologs of X. laevis? Since the level of GAPDH in Figure 1H also looks reduced in Gai2 MO lane, it should be made clear that the apparent knockdown of Gai2 was normalized to GAPDH, rather than being the results of unequal loading of the gel. Yes, I recognize that Figure 1I says normalized, but this is not stated in the results or the methods. Also, was this experiment done with X. laevis or X. tropicalis? I could imagine that if done in X. laevis, the lack of complete knockdown might be due to only one homoeolog being affected.

    R: We appreciate the reviewer comment, and we described in Material and Methods section the region targeted by the morpholino, in both Xenopus species. We added the next paragraph in the Material and Methods section, see page 23 paragraph 1 lines: 7-11.

    “The Gαi2 morpholino (Gαi2MO) was designed as described in the results section to target Gαi2 in both Xenopus species (Xenopus tropicalis and Xenopus laevis). Specifically, it hybridizes with the 5’ UTR of X. tropicalis Gαi2 (NM_203919), 17 nucleotides upstream of the ATG start codon. For X. laevis Gαi2, the morpholino hybridizes with both isoforms described in Xenbase. It specifically targets the 5' UTR of the Gαi2.L isoform (XM_018258962), located 17 nucleotides upstream of the ATG start codon, and the 5' UTR of the Gαi2.S isoform (NM_001097056), situated 275 nucleotides upstream of the ATG.”

    With respect to Figure 1H and 1I, we have specified in the Figure 1 legend that we normalized the data to GAPDH to quantifying the decrease in Gαi2 expression induced by the morpholino.

    See page 37, Figure 1H-I, Legends section.

    1. The knowledge of the efficacy of knockdown in each Xenopus species provided by the information requested in the previous point, would allow the reader to assess the level of knockdown in the remaining assays. To do this, the authors should tell us which assays were done in which species. I am not suggesting that each experiment needs to be done in each species, only that the information should be provided. If the MO is more effective in X. tropicalis - which assays used this species? If the knock down is partial, as shown in Figure 1H-I, which species this represents in the remaining assays would be useful knowledge.

    R: We greatly appreciate the reviewer's valuable comments and suggestions, and as a response, we have incorporated a new supplementary figure (Figure S1). This figure includes a western blot and an in situ hybridization assay illustrating the efficiency of the knockdown in Xenopus laevis. The results presented in Figure S1 demonstrate that the knockdown efficiency is similar in both Xenopus species, allowing for a comparison between Figure 1A-I (X. tropicalis) and Figure 1S (X. laevis).

    To complement this information, we have also improved the section of Material and Methods regarding the experiments in both Xenopus species (Xenopus tropicalis and Xenopus laevis). As detailed in the Materials and Methods section, we employed 20 ng of Gai2MO for Xenopus tropicalis embryos and 35 ng of Gai2MO for Xenopus laevis embryos to deplete cell migration. In both species, in vivo migration was analyzed, resulting in a substantial inhibition of cranial neural crest (NC) migration, ranging from 60% to 80%. Additionally, we conducted dispersion assays in both species. In X.* laevis, in vitro migration was monitored for 10 hours, while in X.** tropicalis, it was tracked for 4 hours, both yielding the same phenotype. We also studied cell morphology and microtubule dynamics in both Xenopus models. However, we used different tracer concentrations for each, with 200 pg for X. laevis and 100 pg for X. tropicalis, as specified in the Materials and Methods section. Our Rac1 and RhoA timelapse experiments were conducted in both species as well, employing pGBD-GFP and rGBD-mCherry probes, respectively, and different probe concentrations as outlined in the Materials and Methods section. These experiments revealed polarity impairment and consistent Rac1 behavior in both Xenopus species. The study of focal adhesion in vivo dynamics using the FAK-GFP tracer was carried out also in both species, resulting in the same phenotype. It is worth noting that the only experiment conducted exclusively in X.** tropicalis* was the focal adhesion disassembly assay with nocodazole.

    Regarding the improvements of the Materials and Method section see page 22, paragraph 2.

    We want to highlight that at the beginning of the Materials and Methods section, we incorporated a paragraph to clarify that “all experiments were conducted in both Xenopus species (X.t and X.l) using distinct concentrations of the morpholino (MO) and mRNA, as specified in each respective methodology description”. This approach consistently yielded similar results. It is important to note that for the figures, we selected the most representative images.

    We have also specified in each figure legend which Xenopus species is depicted.

    Minor comments: While prior studies are referenced appropriately, and the text and figures are mostly clear and accurately presented, the following are a few suggestions that would help the authors improve the presentation of their data and conclusions:

    1. The cell biological experiments convincingly demonstrate that knockdown of Gai2 causes cells to move more slowly. It would be a nice addition to bring the explant experimental data back to the embryo by showing whether the slower moving NC cells in morphants eventually populate the BA. DO they cease to migrate or are they just slower getting to their destination? This could be done by performing snail2 ISH at a later stage (34-35?)

    R: We appreciate the reviewer's insightful point and are currently conducting the in situ hybridization assay at stages 32-36 to address this question. Our plan includes incorporating a supplementary figure showing the results of this assay and integrating this information into both the results and discussion sections.

    1. There are places in the manuscript where the authors use the terms "silencing" or "suppression" of Gai2, when they really mean reduced translation - their system is not a genetic knockout, as clearly demonstrated in Figure 1H-I. I suggest that more accurate wording be used.

    R: We appreciate the reviewer's comment, and we agree that the Gαi2 morpholino impedes Gαi2 translation, leading to a reduction in Gαi2 protein expression. Consequently, we have revised the entire manuscript, replacing the terms “silencing” and “suppression” with “knockdown”.

    1. In Figures 1-5 there are scale of bars on the cell images, but these are not defined in any of the figure legends.

    R: We value the reviewer's comment, and we have revised all the figure legends by including the scale information. Each image has been scaled to 10 µm with varying magnifications.

    1. The abstract is the weakest section of the manuscript, and would have greater impact if it were more clearly written.

    R: We appreciate the reviewer's comment on the abstract, and we have revised and edited it to enhance its quality.

    Abstract:

    “Cell migration is a complex and essential process in various biological contexts, from embryonic development to tissue repair and cancer metastasis. Central to this process are the actin and tubulin cytoskeletons, which control cell morphology, polarity, focal adhesion dynamics, and overall motility in response to diverse chemical and mechanical cues. Despite the well- established involvement of heterotrimeric G proteins in cell migration, the precise underlying mechanism remains elusive, particularly in the context of development.

    This study explores the involvement of Gαi2, a subunit of heterotrimeric G proteins, in cranial neural crest cell migration, a critical event in embryonic development. Our research uncovers the intricate mechanisms underlying Gαi2 influence, revealing its interaction with tubulin and microtubule-associated proteins such as EB1 and EB3, suggesting a regulatory function in microtubule dynamics modulation. Gαi2 knockdown leads to microtubule stabilization, alterations in cell morphology and polarity, increased Rac1-GTP concentration at the leading edge and cell-cell contacts, impaired cortical actin localization and focal adhesion disassembly. Interestingly, RhoA-GTP was found to be reduced at cell-cell contacts and concentrated at the leading edge, providing evidence of Gαi2 significant role in polarity. Remarkably, treatment with nocodazole, a microtubule-depolymerizing agent, effectively reduces Rac1 activity, restoring cranial NC cell morphology, actin distribution, and overall migration. Collectively, our findings shed light on the intricate molecular mechanisms underlying cranial neural crest cell migration and highlight the pivotal role of Gαi2 in orchestrating microtubule dynamics through EB1 and EB3 interaction, modulating Rac1 activity during this crucial developmental process.”

    The molecular regulation of cell movement is a key feature of a number of developmental and homeostatic processes. While many of the proteins involved have been identified, how they interact to provide motility has not been elucidated in any great detail, particularly in embryo-derived cells (as opposed to cell lines). The results obtained from the presented experiments are novel, in-depth and provide a novel paradigm for how G proteins regulate microtubule dynamics which in turn regulate other components of the cytoskeleton required for cell movement. The results will be applicable to many migrating cell types, not just neural crest cells.

    Because of the application of the data to many types of cells that migrate, the audience is expected to include a broad array of developmental biologists, basic cell biologists and those interested in clinically relevant aberrant cell migrations.

    R: We really appreciate the reviewer positive comments and consideration

    From Reviewer 2

    Major comments

    The authors aim to address two issues in this manuscript: a) the role of Gai2 in neural crest development; and b) the mechanism of Gai2 function. While they have done a good job demonstrating a role of Gai2 in NC migration both in vivo and in vitro as well as the effects of Gai2 knockdown on cytoskeleton dynamics, protein distribution of selected polarity and focal adhesion molecules, and Rac1 activation, the link between Gai2 and the downstream effectors is largely correlative. Because of this, the model suggesting the sequential events flowing from Gai2 to microtubule to Rac1 to focal adhesion/actin should be modified to allow room for direct and indirect regulation at potentially multiple entry points.

    R: We appreciate the reviewer's valuable comments. We concur with the reviewer's observation that our experiments do not establish a causal link between Gαi2, EB1/EB3, and Rac1. We established a relationship between Gαi2 and microtubule dynamics (EB1 and EB3) to regulate Rac1 polarity through co-immunoprecipitation assays, which reveal protein interactions within an interactor complex. Therefore, while our findings support the involvement of Gαi2 in coordinating cranial NC cell migration alongside EB1, EB3, and Rac1, we cannot exclude the possibility that this regulation may occur through other intermediary proteins, such as GEFs, GAPs, GDIs, and others. As a result, we have revised our model and its description in accordance with the reviewer suggestion.

    We have edited the discussion/conclusion, model and the legend at Figure 6. See page 16 (paragraph 2, last line), 17 (paragraph 1, last line), 22 (paragraph 1, last line 17-20), 42 (Legend Fig. 6).

    Specific major comments are as the following: Strengths: -Determination of a role of Gai2 in neural crest migration is novel. -The effect of Gai2 knockdown on membrane protrusion morphology and microtubule stability and dynamics are demonstrated nicely. -Quantification of experimental perimeters has been performed throughout the manuscript in all the figures, and statistical analysis is included in the figures.

    R: We appreciate the reviewer positive comments

    Weaknesses: -The heavy focus of the study on microtubule is due to the previous publication on the function of Gai2 in regulation of microtubule during asymmetrical cell division. However, the activity of Gai2 is likely cell type-specific, as it has not been shown to control microtubule during cytokinesis in general. It is equally likely that Gai2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. The tone of the discussion should therefore be softened.

    R: We greatly appreciate and agree with the comment from the reviewer, highlighting the possibility that Gαi2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. In this regard, we have revised our manuscript to include a discussion of this point. We added the next paragraph in the Discussion/Conclusion section, page 20-21.

    “It is well established that the activity from the Rho family of small GTPases is controlling cytoskeletal organization during migration (Ridley et al., 2015). Contrariwise, it has been described in many cell types, that microtubules dynamic polymerization plays a crucial role in establishing the structural foundation for cell polarization, consequently influencing the direction of cell motility (Watanabe et al., 2005). Our results appear to align with this latter view. While it is reasonable to postulate the possibility that Gαi2 regulates Rac1 activity, subsequently influencing actin and microtubule dynamics, our findings in the context of cranial NC cells, lend support to an alternative sequence of events. Initially, Gαi2 knockdown leads to a decrease in microtubule dynamics, which in turn increase Rac-GTP towards the leading edge. This shift is accompanied by reduced levels of cortical actin and impaired focal adhesion disassembly, culminating in compromised cell migration. Notably, nocodazole, a microtubule-depolymerizing agent, not only diminishes Rac-GTP localization at the leading edge but also rescues cell morphology, restores normal cortical actin localization, and promotes focal adhesion disassembly, thereby facilitating cell migration. If Rac1 activity were indeed upstream of microtubules, it would be expected that nocodazole would not reduce Rac-GTP levels at the cell leading edge. These results suggest that the regulation of Rac1 activity may follow, rather than precede, alterations in microtubule dynamics, in the context of NC cells. Furthermore, in support of our model, our protein interaction analysis demonstrates Gαi2 interacting with microtubule components such as EB proteins and tubulin. As we already mention above, earlier studies have reported that microtubule dynamics promote Rac1 signaling at the leading edge and by releasing RhoGEFs promote RhoA signaling as well (Best et al., 1996; Garcin and Straube, 2019; Moore et al., 2013; Waterman-Storer et al., 1999). In addition, it is well-documented that RhoGEFs interact with microtubules, including bPix, a GEF for Rac1 and Cdc42, which, in turn, promotes tubulin acetylation (Kwon et al., 2020). Interestingly, in ovarian cancer cells, Gαi2 has been shown to activate Rac1 through an interaction with bPix, thereby jointly regulating migration in response to LPA (Ward et al., 2015). Taken together, these findings further support our proposed model (refer to Fig. 6).”

    -The effect of rescue of NC migration with Rac1 inhibitor is marginal and the result is hard to interpret considering the inhibitor also blocks control NC migration. Either lower doses of Rac1 inhibitor can be used or the experiment can be removed from the manuscript, as Rac1 is required for membrane protrusions and the inhibitor doses can be hard to titrate.

    __ __R: We appreciate and agree with the reviewer's comments. To address this concern and enhance clarity, we have incorporated the following paragraph into the manuscript within the Discussion section. Additionally, we have included information on the range of NSC23766 concentrations used for this analysis in the Materials and Methods section. Page 24, Explants and microdissection.

    “It is worth noting that we conducted Rac inhibitor NSC23766 trials at concentrations ranging from 20 nM to 50 nM for X. laevis and between 10 nM to 30 nM for X. tropicalis. In both cases, higher concentrations of the Rac inhibitor proved to be lethal, underscoring the essential role of Rac1 in both cell migration and cell survival. Specifically, the described concentrations of 20 nM for X. laevis and 10 nM for X. tropicalis led to a partial rescue of the observed phenotype. This suggests that these concentrations are sufficient to demonstrate that the increase in Rac1-GTP resulting from Gαi2 morpholino knockdown impairs cell migration. The partial rescue can be attributed to the crucial role of microtubule dynamics in cell migration, which acts upstream of Rac activity. Additionally, Rac is pivotal for the modulation of cell polarity at the leading edge of migration. It is worth emphasizing that Rac1 levels are critical for cell migration, as demonstrated by other researchers. Lower concentrations of Rac1-GTP have been shown to hinder cell migration in cells deficient in Rac1, leading to a significant reduction in wound closure and random cell migration (Steffen et al., 2013).

    Therefore, we believe that the lower concentration of NSC23766 used in our assay was adequate to reduce the abnormal Rac1-GTP activity in the morphant NC cells. However, it is important to note that for normal NC cell, this level of reduction in Rac1-GTP activity is critical and sufficient to impair normal migration”.

    See page 12, paragraph 2, lines 8-11, 14-16, 23-25.

    Steffen A, Ladwein M, Dimchev GA, Hein A, Schwenkmezger L, Arens S, Ladwein KI, Margit Holleboom J, Schur F, Victor Small J, Schwarz J, Gerhard R, Faix J, Stradal TE, Brakebusch C, Rottner K. Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation. J Cell Sci. 2013 Oct 15;126(Pt 20):4572-88. doi: 10.1242/jcs.118232. Epub 2013 Jul 31. PMID: 23902686; PMCID: PMC3817791.

    -Since the defects seem to result partially from the inability of the NC cells to retract and move away, it may help to either include some data on Rho activation patterns in knockdown cells or simply add some discussion about the issue.

    R: We acknowledge and sincerely appreciate the reviewer's valuable comments on this pivotal aspect, which significantly enhances our capacity to elucidate the impact of Gαi2 knockdown on cell polarity. To address this crucial point, we have introduced an experiment that examines RhoA-GTP localization under Gαi2 knockdown conditions, and we have incorporated a supplementary figure S3 into our manuscript. This newly added figure clearly demonstrates that, under Gαi2 knockdown conditions, and in contrast to control cells, RhoA-GTP localization is substantially disrupted at cell-cell contacts and now detected at the leading edge of the cell, providing compelling evidence of cell polarity defects (refer to Figure S3). In response to these results, we have included a description of these findings in the Results section (please see page 11-12) and a dedicated paragraph in the Discussion section (please see page 18, paragraph 2, line 15-16, page 19, paragraph 1, lines 6-12).

    Results section 1: “To achieve this, we explored whether Gαi2 regulates the subcellular distribution of active Rac1 and RhoA in cranial NC explants under Gαi2 loss-of-function conditions, considering their pivotal roles in cranial NC migration and contact inhibition of locomotion (CIL) (Carmona-Fontaine et al., 2011; Moore et al., 2013; Leal et al., 2018). Hence, we employed mRNA encoding the small GTPase-based probe, enabling specific visualization of the GTP-bound states of these proteins.”

    Results section 2: “Consistent with earlier observations by Carmona-Fontaine et al. (2011), in control cranial NC cells, active Rac1 displayed prominent localization at the leading edge of migrating cells, whereas its presence was reduced at cell-cell contacts, coincident with an increase in RhoA-GTP levels (white arrows in Fig. 4A, supplementary material Figure S3A). On the contrary, in comparison to the control cells, Gαi2 morphants exhibit a pronounced accumulation of active Rac1 protein in the protrusions at cell-cell contacts, where active RhoA localization is conventionally expected (white arrow in Fig. 4B, supplementary material Figure S3A and movie S6). In contrast to control cells, a notable shift in the localization of active RhoA protein was observed, with its predominant accumulation now detected at the leading edge of the cell, instead of the typical localization towards the trailing edge or cell-cell contacts (__supplementary material Figure S3B). __These findings suggest a dysregulation of contractile forces that align with the observed distribution of active RhoA, cortical actin disruption, and diminished retraction in cell treated with Gαi2MO.”

    Discussion section:

    “Other studies have reported that microtubule assembly promotes Rac1 signaling at the leading edge, while microtubule depolymerization stimulates RhoA signaling through guanine nucleotide exchange factors associated with microtubule-binding proteins controlling cell contractility, via Rho-ROCK and focal adhesion formation (Krendel et al., 2002; Ren et al., 1999; Best et al., 1996; Garcin and Straube, 2019; Waterman-Storer et al., 1999; Bershadsky et al., 1996; Moore et al., 2013). This mechanism would contribute to establishing the antero-posterior polarity of cells, crucial for maintaining migration directionality, underscoring the significance of regulating microtubule dynamics in directed cell migration. These findings closely align with the results obtained in this investigation, demonstrating that Gαi2 loss of function reduces microtubule catastrophes and promotes tubulin stabilization, resulting in increased localization of active Rac1 at the leading edge and cell-cell contacts, while decreasing active RhoA at the cell-cell contact but increasing it at the leading edge. This possibly reinforces focal adhesion, which is consistent with the presence of large and highly stable focal adhesions under Gαi2 knockdown conditions. This finding also suggests a dysregulation of contractile forces in comparison to control cells, a result that aligns with the observed distribution of active RhoA, cortical actin distribution and diminished retraction in cells treated with Gαi2MO. This strikingly contrasts with the normal cranial NC migration phenotype, where Rac1 is suppressed while active RhoA is increased at cell-cell contacts during CIL, leading to a shift in polarity towards the cell-free edge to sustain directed migration (Theveneau et al., 2010; Shoval and Kalcheim, 2012; Leal et al., 2018).”

    -To consider focal adhesion dynamics, live imaging should be used in the analysis. The fixed samples are different from each other, and natural variations of focal adhesion may exist among the samples. This can obscure data collection and quantification.

    R: We agree with the reviewer that focal adhesion (FA) dynamics need to be analysed using live imaging. Indeed, Fig 5E-H shows an extensive analysis of FA using live imaging of neural crest expressing FAK-GFP. As complement to this live imaging analysis, and in order to analyse the effect on the endogenous levels of FA proteins, we performed immunostaining against FA. Both experiments using live imaging or fixed cells produce similar results, and they are consistent with our model on the role of Gαi2 on FA dynamics.

    Minor comments -Fig. 2, the centrosomes in control cells are not always obvious. The microtubules simply seem to be more networked and more fluid in control cells. This should be clarified with either marking the centrosomes in the figure or modifying the wording in the manuscript.

    R: We appreciate and concur with the reviewer's comment on this matter. As pointed out by the reviewer, the precise localization of the centrosome is not consistently clear in all cells. In response to this observation, we have revised the manuscript to emphasize this aspect solely as “microtubule morphology”. Please refer to the Results section description Figure 2.

    -In Fig. 3, a better negative control for co-IP should be using anti-V5 antibody to IP against tubulin/EB1/EB3 in the absence of Gai2-V5.

    R: We appreciate the reviewer's comment, and we agree about the controls that the reviewer suggest. We can inform that we have done by triplicate all the Co-IPP. Although, if is necessary we will do the controls suggested. We present this assay as a plan.

    -The data for cell polarity proteins Par3 and PKC-zeta seem to be out of place. It is unclear whether mis-localization of these proteins has anything to do with NC migration defects induced by Gai2 knockdown. The conclusion does not seem to be affected if the data are taken out of the manuscript.

    R: We appreciate the reviewer's concern, and we would like to highlight two points in this regard. Firstly, we have included these results as additional data to support the impact of Gai2 knockdown on cell polarity, given that these two proteins are commonly used polarity markers. Secondly, we have discussed this aspect extensively in the Discussion section of the manuscript. (See page 19, paragraph 1, lines 18-28)

    In that section, we delve into the relationship between aPKC, Par3, and Gαi2 in controlling cell polarity during asymmetric cell division, as described in Hao et al., 2010. Par3 is known to play a role in regulating microtubule dynamics and Rac1 activation through its interaction with Rac-GEF Tiam1 (Chen et al., 2005). Additionally, it has been shown to promote microtubule catastrophes and inhibit Rac1/Trio signaling, regulating Contact Inhibition of Locomotion (CIL) as demonstrated in Moore et al., 2013. Thus, we believe that the data we present support the relationship between Par3 and aPKC localization changes and the neural crest migration defects induced by Gαi2 knockdown, probably by controlling microtubule dynamics. However, we have moved these results as part of the supplementary Figure S3.

    -In Suppl. Fig. 1, protrusion versus retraction should be defined more clearly. The retraction shown in this figure seems to be just membrane between protrusions instead of actively retracting membrane.

    R: We appreciate the reviewer's comments, and here we aim to provide a clearer description of our approach to this analysis. For the measurement of protrusion extension/retraction, we conducted two distinct experiments. The first, as described in Figure 1, involved measuring membrane extension and retraction in live cell using membrane-GFP by utilizing the image subtraction tool in ImageJ, which highlights changes in the membrane in red. Secondly, we employed ADAPT software to quantify cell perimeter based on fluorescence intensity in live cell using lifeactin-GFP, distinguishing membrane extension in green and retraction in red (as has been shown similarly in Barry et al., 2015). In both approaches, we observed a substantial increase in membrane protrusion (both in area and extension) and protrusion stability in Gαi2 morphants. Hence, we have revised the Materials and Methods section of the manuscript and included this clarification.

    See Materials and Methods section, Cell dispersion and morphology, page 25-26.

    Barry DJ, Durkin CH, Abella JV, Way M. Open source software for quantification of cell migration, protrusions and fluorescence intensities. J Cell Biol. 2015. Doi: 10.1083/jcb.201501081

    -Discussion can be improved by better incorporating all the components to make a cohesive story on how Gai2 works to regulate migration in the context of the neural crest cells.

    R: We appreciate the reviewer's comment and agree. To enhance the manuscript, we have included a new paragraph at the end of the Discussion/Conclusion section specifically addressing this point. For more details, please refer to page 21-22.

    “In the context of collective cranial NC cells migration, our findings reveal the pivotal role played by Gαi2 in orchestrating the intricate interplay of microtubule dynamics and cellular polarity. When Gαi2 levels are diminished, we observe significant impediments in the ability of cells to efficiently navigate through their environment, resulting in a range of distinct effects. First and foremost, Gαi2 deficiency leads to the diminished ability of cells to adjust and reorient new protrusions effectively. Primary protrusions exhibit higher stability and heightened levels of active Rac1/RhoA when compared to control conditions in the leading edge. In addition, we observe a notable increase in protrusion area, a decrease in retraction velocity, and an enhanced level of cell-matrix adhesion in Gαi2 knockdown cells. These findings underscore the pivotal role that Gαi2 plays in the modulation of various cellular dynamics essential for collective cranial NC cells migration. Notably, the application of nocodazole, a microtubule-depolymerizing agent, and NSC73266, a Rac1 inhibitor, to Gαi2 knockdown cells leads to the rescue of the observed effects, thus facilitating migration. This observed response closely mirrors the outcomes associated with Par3, a known regulator of microtubule catastrophe during contact inhibition of locomotion (CIL) in NC cells. This parallel implies that there exists a delicate equilibrium between microtubule dynamics and Rac1-GTP levels, crucial for the establishment of proper cell polarity during collective migration. Our findings collectively position Gαi2 as a central master regulator within the intricate framework of collective cranial NC migration. This master regulator's role is pivotal in orchestrating the dynamics of polarity, morphology, and cell-matrix adhesion by modulating microtubule dynamics through interactions with EB1 and EB3 proteins, possible in a protein complex involving other intermediary proteins such as GDIs, GAPs and GEFs, thus fostering crosstalk between the actin and tubulin cytoskeletons. This orchestration ultimately ensures the effective collective migration of cranial NC cells (Fig. 6).”

    From Reviewer #3

    Major comments:

    1. The authors focus exclusively on the analysis of the subcellular levels of Rac1, which is probably related to the fact that they observe large extended protrusions with high Rac1 activity. However, as the authors note, a global fine-tuning of Rho GTPase activity is required for neural crest migration. One of the observed phenotypes of Gαi2-morphant neural crest cells is a decrease in cell dispersion, which may be caused by defects in contact inhibition of locomotion (CIL). This process involves a local activation of RhoA at cell-cell contact sites (Carmona-Fontaine et al., 2008). Furthermore, in fibroblast, RhoA/ROCK activity is required for the front-rear polarity switch during CIL (Kadir et al., 2011). Interestingly, similar to the Gαi2 loss of function phenotype, ROCK inhibition leads to microtubule stabilization, which can be rescued by nocodazole treatment, restoring microtubule dynamics and CIL. Therefore, it would also be interesting to know how RhoA activity is affected in Gαi2-morphant NC cells. At a minimum, this point should be be included in the discussion.

    R: We acknowledge and sincerely appreciate the reviewer's valuable comments on this pivotal aspect, which significantly enhances our capacity to elucidate the impact of Gαi2 knockdown on cell polarity. To address this crucial point, we have introduced an experiment that examines RhoA-GTP localization under Gαi2 knockdown conditions, and we have incorporated a supplementary figure S3 into our manuscript. This newly added figure clearly demonstrates that, under Gαi2 knockdown conditions and in contrast to control cells, RhoA-GTP localization is substantially disrupted at cell-cell contacts and now detected at the leading edge of the cell, providing compelling evidence of cell polarity defects (refer to Figure S2). In response to these results, we have included a description of these findings in the Results section (please see page 11-12) and a dedicated paragraph in the Discussion section (please see page 18, paragraph 2, line 15-16, page 19, paragraph 1, lines 6-12).

    Results section 1: “To achieve this, we explored whether Gαi2 regulates the subcellular distribution of active Rac1 and RhoA in cranial NC explants under Gαi2 loss-of-function conditions, considering their pivotal roles in cranial NC migration and contact inhibition of locomotion (CIL) (Carmona-Fontaine et al., 2011; Moore et al., 2013; Leal et al., 2018). Hence, we employed mRNA encoding the small GTPase-based probe, enabling specific visualization of the GTP-bound states of these proteins.”

    Results section 2: “Consistent with earlier observations by Carmona-Fontaine et al. (2011), in control cranial NC cells, active Rac1 displayed prominent localization at the leading edge of migrating cells, whereas its presence was reduced at cell-cell contacts, coincident with a increase in RhoA-GTP levels (white arrows in Fig. 4A, supplementary material Figure S2A). On the contrary, in comparison to the control cells, Gαi2 morphants exhibit a pronounced accumulation of active Rac1 protein in the protrusions at cell-cell contacts, where active RhoA localization is conventionally expected (white arrow in Fig. 4B, supplementary material Figure S3B and movie S6). In contrast to control cells, a notable shift in the localization of active RhoA protein was observed, with its predominant accumulation now detected at the leading edge of the cell, instead of the typical localization towards the trailing edge or cell-cell contacts (__supplementary material Figure S2). __These findings suggest a dysregulation of contractile forces that align with the observed distribution of active RhoA, cortical actin disruption, and diminished retraction in cell treated with Gαi2MO.”

    Discussion section:

    “Other studies have reported that microtubule assembly promotes Rac1 signaling at the leading edge, while microtubule depolymerization stimulates RhoA signaling through guanine nucleotide exchange factors associated with microtubule-binding proteins controlling cell contractility, via Rho-ROCK (cita) and focal adhesion formation (Krendel et al., 2002; Ren et al., 1999; Best et al., 1996; Garcin and Straube, 2019; Waterman-Storer et al., 1999; Bershadsky et al., 1996; Moore et al., 2013). This mechanism would contribute to establishing the antero-posterior polarity of cells, crucial for maintaining migration directionality, underscoring the significance of regulating microtubule dynamics in directed cell migration. These findings closely align with the results obtained in this investigation, demonstrating that Gαi2 loss of function reduces microtubule catastrophes and promotes tubulin stabilization, resulting in increased localization of active Rac1 at the leading edge and cell-cell contacts and decreasing active RhoA at the cell-cell contact but increasing at the leading edge, possibly reinforcing focal adhesion, which align with our result here that show large and highly stable focal adhesions under Gαi2 knockdown conditions. This finding also suggests a dysregulation of contractile forces in comparison to control cells, a result that aligns with the observed distribution of Active RhoA, cortical actin distribution and diminished retraction in cells treated with Gαi2MO. This strikingly contrasts with the normal cranial NC migration phenotype, where Rac1 is suppressed while active RhoA is increased at cell-cell contacts during CIL, leading to a shift in polarity towards the cell-free edge to sustain directed migration (Theveneau et al., 2010; Shoval and Kalcheim, 2012; Leal et al., 2018).”

    The co-Immunoprecipitation data lack marker bands (larger images/sections of the blots would be preferable) and the labelling is not clear. What do the white arrows in Fig. 3H,I mean? What does "elu" and "non eluted" mean? Did the reverse IP work as well?

    R: We appreciate the reviewer's comments, and here we intend to provide a more detailed explanation of our approach to this analysis. Since we do not possess a secondary antibody specific to the heavy chain, our method involves eluting the co-immunoprecipitated proteins to visualize those with weights close to that of the light chain (such as EB1). We have outlined this elution step in the “Cell lysates and co-immunoprecipitation” protocol in the Materials and Methods section. To ensure proper control, we load both fractions - the eluted (or supernatant) and non-eluted (or resin) fractions - to monitor the amount of protein extracted from the resin using a 1% SDS solution. It's important to note that the elution step, as indicated by the V5 signal, is not entirely efficient, and a significant portion of the protein remains bound to the resin. This issue may also apply to the EB1 protein; however, it is still possible to visualize both bands (Gαi2V5 and EB1).

    We have revised the legend for Figure 3 to include an explanation of the terms 'elu' (eluted fraction) and 'non-eluted' (non-eluted fraction). We have also included the explanation of the white arrows’ significance in the legends for Figure 3H and 3I. These arrows indicate the bands corresponding to the immunoprecipitated proteins.

    We also agree with the reviewer’s suggestion to conduct the reverse IP. We can inform that we have done by triplicate all the Co-IPP. Although, if is necessary we will do the controls suggested. We present this assay as a plan.

    The presentation of the Delaunay triangulations varies in quality. In Fig. 1 J/K the cells are clearly visible in the images, while this is not the case in Fig. 3 J-M and Fig. 4K-N. Conversely, the Delaunay triangulations in Fig. 1L are mainly black, while they are clear in Fig. 3 and 4. Perhaps the authors could find a more consistent way to present the data. Were the explants all approximately the same size at the beginning of the experiment? The Gαi2-morphant explant in Fig. 3K appears to be unusually small.

    R: We appreciate the reviewer’s concerns and have taken steps to address them. To improve the quality of our data, we have made enhancements to the presentation of Figures 3 (panels J-M) and Figure 4 (panels K-N). Specifically, we have standardized the Delaunay triangulation representations.

    Regarding the size of the explants at the beginning of the experiments, they were indeed approximately similar in size. We confirmed this by including a reference point (point 0) for each condition in the figures 3. However, in the panels presented, we show the results after 10 hours (Figure 3, X. laevis) and 4 hours (Figure 4, X. tropicalis) to assess cell dispersion, as indicated in the respective figure legends. This uniformity in size was further ensured by the calculation used to quantify dispersion. For the dispersion assay, we normalized each initial size of the explant upon the control, and we have added another representative explant of Gαi2 morpholino with its Delaunay triangulation to facilitate the experiment interpretation. Every Delaunay triangulation calculates the area generated between three adjacent cells and it grows depending on how much disperse are the cells between each other in the final point. (See Material and Methods section, Cell dispersion and morphology). As we can see in the manuscript, in every dispersion experiment that we have performed with Gαi2 morpholino, the cells cannot disperse at all. Furthermore, to analyze the dispersion rate in this experiment we use Control n= 21 explants, Gαi2MO n= 24 explants, Gαi2MO + 65 nM Nocodazole n= 36 explants, Control + 65 nM Nocodazole n= 30 explants (as we mentioned in the manuscript legend).

    Why was the tubulin distribution in Fig. 2F measured from the nucleus to the cell cortex? Would it not make more sense to include cell protrusions? This does not seem to be the case in the example shown in Fig. 2F.

    R: We appreciate the reviewer's concern. We would like to clarify that for the tubulin distribution measurements, we indeed measured from the nucleus to the cell protrusion. As a result, we have made an edit to Figure 2 (panel 2F) to provide further clarity on this matter.

    The immunostaining for acetylated tubulin (Fig. 3A,B) looks potentially unspecific and seems to co-localize with actin (for comparison see Bance et al., 2019). For imaging and quantification, it may be better to use tubulin co-staining to calculate the percentage of acetylated tubulin. Please also add marker bands to the Western blot in Fig. 3C. If this issue cannot be resolved it may be better to only include the Western blot data.

    R: We appreciate the reviewer's concern about the potential unspecific nature of acetylated-tubulin and its co-localization with actin, particularly in Figure 3. Regarding the co-localization with actin, it is predominantly observed in panel B, and we attribute this phenomenon to the Gαi2 morphant phenotype, where cortical actin is notably reduced, creating the appearance of co-localization. However, we will assess the experiment as suggested by the reviewer. Therefore, our plan is to conduct an immunostaining for acetylated tubulin and tubulin in both control and Gαi2 knockdown conditions. This will allow us to calculate the percentage of acetylated tubulin and complement the western blot analysis.

    We have included marker weight indications on the western blot panel in Figure 3C.

    The model in Fig.6 indicates that Gαi2 inhibits EB1/3. What is the experimental evidence and the proposed mechanism for this? In the discussion, the authors cite evidence that Gαi activates the intrinsic GTPase activity of tubulin, which would destabilize microtubules by removing the GTP cap. However, this mechanism would not directly affect EB1 and EB3 stability as the Fig. 6A seems to suggest. The authors also mention that EB3 appears to be permanently associated with microtubules in Gαi2-morphant cells. How would this work, given that end-binding proteins bind to the cap region? Are the authors suggesting that there is an extended cap region in Gαi2 morphants?

    R: We appreciate the reviewer's valuable comments. We agree with the reviewer's observation that our experiments do not establish a causal link between Gαi2, EB1/EB3, and Rac1. We established a relationship between Gαi2 and microtubule dynamics (EB1 and EB3) to regulate Rac1 polarity through co-immunoprecipitation assays, which reveal protein interactions within an interactor complex. In addition, in Gαi2 Knockdown conditions we have found a strong reduction in microtubules dynamics following EB-GFP comets. Regarding the observation that EB3 seems to be persistently associated with microtubules in Gαi2-morphant cells, we wish to clarify that this is a speculation based on the microtubule phenotype observed during our dynamic analysis, where they appear more like lines rather than comets. It is important to note that none of the experiments conducted in this study conclusively demonstrate this, and thus, it remains a suggestion. Therefore, while our findings support the involvement of Gαi2 in coordinating cranial NC cell migration alongside EB1, EB3, and Rac1, we cannot exclude the possibility that this regulation may occur through other intermediary proteins, such as GEFs, GAPs, GDIs, and others. As a result, we have revised our model in accordance with the reviewer suggestion.

    We have edited both the model and the legend at Figure 6. Gαi2 controls cranial NC migration by regulating microtubules dynamics.

    Considering this, we have reviewed the manuscript to provide clarity on this point. See page 16 (paragraph 2, last line), 17 (paragraph 1, last line), 22 (paragraph 1, last line 17-20), 42 (Legend Fig. 6).

    Minor comments

    1. The citation of Wang et al. 2018 in the introduction does not seem to fit.

    R: We appreciate the correction provided by the reviewer. We have carefully reviewed the Introduction and Reference sections and have corrected this error.

    2.Does the graph in Fig. 4S show average values for the three conditions? If so, what is the standard deviation?

    R: We appreciate the reviewer’s concern and we have added the standard deviation to Figure 4S.

    3.From the images in Fig. 2G and H, it is difficult to understand what the difference is between the four groups shown.

    R: We appreciate the reviewer's comment, and to clarify this point, we would like to explain that the comparison has been made between each type of comet. The PlusTipTracker software separates comets based on their speed and lifetime, classifying them as fast long-lived, fast short-lived, slow long-lived, or slow short-lived. In both conditions (control and morphant cells), we compared the percentage of each type of comet, as previously described in Moore et al., 2013. The results demonstrate that morphant cells exhibit an increase in slow comets compared to control cells. The same explanation is described in the Material and Methods section on page 26, Microtubule dynamics analysis.

    Reviewer #3 (Significance (Required)): Overall, the study is well executed and significantly advances our understanding of the control and role of microtubule dynamics in cell migration, which is much less understood compared to the function of the actin cytoskeleton in this process. The strength of the study is the use of state-of-the-art (live imaging) techniques to characterize the role of Gαi in neural crest migration at the cellular/subcellular level. This article will be of interest to a broad readership, including researchers interested in basic embryonic morphogenesis, cell migration, and cytoskeletal dynamics, as well as translational/clinical researchers interested in cancer progression or wound healing.

    R: We really appreciate the reviewer positive comments and consideration. We believe that the review process has significantly strengthened our manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: The manuscript by Villaseca et al. analyzes the role of Gαi2 in cranial neural crest migration and reveals a novel mechanistic link to microtubule dynamics. The authors nicely demonstrate that Gαi2 is required for Xenopus neural crest migration and affects cell dispersion, cell polarity, focal adhesion turnover, and microtubule dynamics. They find that Gαi2-morphant neural crest cells are elongated, have larger, more stable protrusions, higher active Rac1 levels, and a concentration of microtubules at the leading edge. Using co-immunoprecipitation, the authors show that Gαi2 forms a complex with α-tubulin and the microtubule plus-end binding proteins EB1 and EB3, which are known regulators of microtubule dynamics. Time-lapse imaging shows that Gαi2 loss of function increases microtubule stability, which is further supported by an increase in acetylated tubulin levels. Consistently, treatment with nocodazole, which inhibits microtubule polymerization, as well as treatment with a Rac1 inhibitor, is able to rescue cell dispersion and morphology of Gαi2-morphant neural crest cells. The authors propose a model, whereby Gαi2 interacts with components of the plus-tip microtubule-binding complex to control microtubule dynamics and Rac1 activity to establish cell polarity, disassemble focal adhesion, and thereby facilitate neural crest migration.

    Major comments:

    1. The authors focus exclusively on the analysis of the subcellular levels of Rac1, which is probably related to the fact that they observe large extended protrusions with high Rac1 activity. However, as the authors note, a global fine-tuning of Rho GTPase activity is required for neural crest migration. One of the observed phenotypes of Gαi2-morphant neural crest cells is a decrease in cell dispersion, which may be caused by defects in contact inhibition of locomotion (CIL). This process involves a local activation of RhoA at cell-cell contact sites (Carmona-Fontaine et al., 2008). Furthermore, in fibroblast, RhoA/ROCK activity is required for the front-rear polarity switch during CIL (Kadir et al., 2011). Interestingly, similar to the Gαi2 loss of function phenotype, ROCK inhibition leads to microtubule stabilization, which can be rescued by nocodazole treatment, restoring microtubule dynamics and CIL. Therefore, it would also be interesting to know how RhoA activity is affected in Gαi2-morphant NC cells. At a minimum, this point should be be included in the discussion.
    2. The co-Immunoprecipitation data lack marker bands (larger images/sections of the blots would be preferable) and the labelling is not clear. What do the white arrows in Fig. 3H,I mean? What does "elu" and "non eluted" mean? Did the reverse IP work as well?
    3. The presentation of the Delaunay triangulations varies in quality. In Fig. 1 J/K the cells are clearly visible in the images, while this is not the case in Fig. 3 J-M and Fig. 4K-N. Conversely, the Delaunay triangulations in Fig. 1L are mainly black, while they are clear in Fig. 3 and 4. Perhaps the authors could find a more consistent way to present the data. Were the explants all approximately the same size at the beginning of the experiment? The Gαi2-morphant explant in Fig. 3K appears to be unusually small.
    4. Why was the tubulin distribution in Fig. 2F measured from the nucleus to the cell cortex? Would it not make more sense to include cell protrusions? This does not seem to be the case in the example shown in Fig. 2F.
    5. The immunostaining for acetylated tubulin (Fig. 3A,B) looks potentially unspecific and seems to co-localize with actin (for comparison see Bance et al., 2019). For imaging and quantification, it may be better to use tubulin co-staining to calculate the percentage of acetylated tubulin. Please also add marker bands to the Western blot in Fig. 3C. If this issue cannot be resolved it may be better to only include the Western blot data.
    6. The model in Fig.6 indicates that Gαi2 inhibits EB1/3. What is the experimental evidence and the proposed mechanism for this? In the discussion, the authors cite evidence that Gαi activates the intrinsic GTPase activity of tubulin, which would destabilize microtubules by removing the GTP cap. However, this mechanism would not directly affect EB1 and EB3 stability as the Fig. 6A seems to suggest. The authors also mention that EB3 appears to be permanently associated with microtubules in Gαi2-morphant cells. How would this work, given that end-binding proteins bind to the cap region? Are the authors suggesting that there is an extended cap region in Gαi2 morphants?

    Minor comments

    1. The citation of Wang et al. 2018 in the introduction does not seem to fit.
    2. Does the graph in Fig. 4S show average values for the three conditions? If so, what is the standard deviation?
    3. From the images in Fig. 2G and H, it is difficult to understand what the difference is between the four groups shown.

    Referees cross-commenting The concerns raised by my colleagues are entirely valid.

    Significance

    Overall, the study is well executed and significantly advances our understanding of the control and role of microtubule dynamics in cell migration, which is much less understood compared to the function of the actin cytoskeleton in this process. The strength of the study is the use of state-of-the-art (live imaging) techniques to characterize the role of Gαi in neural crest migration at the cellular/subcellular level. This article will be of interest to a broad readership, including researchers interested in basic embryonic morphogenesis, cell migration, and cytoskeletal dynamics, as well as translational/clinical researchers interested in cancer progression or wound healing.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    The manuscript by Villaseca et al. describes functional analysis of Gai2 in cranial neural crest (CNC) migration using the frog Xenopus as their model system. The authors performed the loss-of-function assay to knock down expression of endogenous of Gai2 and discovered that CNC migration was impaired in the absence of changes of CNC fate specification. Based on the literature on Gai2 activities in other cellular contexts, the authors speculated that Gai2 might regulate microtubule dynamics and Rac1 function. Their studies using immunofluorescence (IF) and live-cell imaging indeed showed that microtubules were stabilized in membrane protrusions with concurrent activation of Rac1 in Gai2 knockdown cells. In addition, focal adhesion turnover was reduced. They further demonstrated that the CNC migration defects could be partially rescued by destabilization of microtubules with chemical treatment. The authors conclude from the studies that Gai2 orchestrates microtubule dynamics and modulates Rac1 activation during neural crest migration.

    Major comments

    The authors aim to address two issues in this manuscript: a) the role of Gai2 in neural crest development; and b) the mechanism of Gai2 function. While they have done a good job demonstrating a role of Gai2 in NC migration both in vivo and in vitro as well as the effects of Gai2 knockdown on cytoskeleton dynamics, protein distribution of selected polarity and focal adhesion molecules, and Rac1 activation, the link between Gai2 and the downstream effectors is largely correlative. Because of this, the model suggesting the sequential events flowing from Gai2 to microtubule to Rac1 to focal adhesion/actin should be modified to allow room for direct and indirect regulation at potentially multiple entry points.

    Specific major comments are as the following:

    Strengths:

    -Determination of a role of Gai2 in neural crest migration is novel. -The effect of Gai2 knockdown on membrane protrusion morphology and microtubule stability and dynamics are demonstrated nicely. -Quantification of experimental perimeters has been performed throughout the manuscript in all the figures, and statistical analysis is included in the figures.

    Weaknesses:

    • The heavy focus of the study on microtubule is due to the previous publication on the function of Gai2 in regulation of microtubule during asymmetrical cell division. However, the activity of Gai2 is likely cell type-specific, as it has not been shown to control microtubule during cytokinesis in general. It is equally likely that Gai2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. The tone of the discussion should therefore be softened.
    • The effect of rescue of NC migration with Rac1 inhibitor is marginal and the result is hard to interpret considering the inhibitor also blocks control NC migration. Either lower doses of Rac1 inhibitor can be used or the experiment can be removed from the manuscript, as Rac1 is required for membrane protrusions and the inhibitor doses can be hard to titrate.
    • Since the defects seem to result partially from the inability of the NC cells to retract and move away, it may help to either include some data on Rho activation patterns in knockdown cells or simply add some discussion about the issue.
    • To consider focal adhesion dynamics, live imaging should be used in the analysis. The fixed samples are different from each other, and natural variations of focal adhesion may exist among the samples. This can obscure data collection and quantification.

    Minor comments

    • Fig. 2, the centrosomes in control cells are not always obvious. The microtubules simply seem to be more networked and more fluid in control cells. This should be clarified with either marking the centrosomes in the figure or modifying the wording in the manuscript.
    • In Fig. 3, a better negative control for co-IP should be using anti-V5 antibody to IP against tubulin/EB1/EB3 in the absence of Gai2-V5.
    • The data for cell polarity proteins Par3 and PKC-zeta seem to be out of place. It is unclear whether mis-localization of these proteins has anything to do with NC migration defects induced by Gai2 knockdown. The conclusion does not seem to be affected if the data are taken out of the manuscript.
    • In Suppl. Fig. 1, protrusion versus retraction should be defined more clearly. The retraction shown in this figure seems to be just membrane between protrusions instead of actively retracting membrane.
    • Discussion can be improved by better incorporating all the components to make a cohesive story on how Gai2 works to regulate migration in the context of the neural crest cells.

    Referees cross-commenting I agree with other reviewers' comments.

    Significance

    The authors demonstrate a role of Gai2 in regulation of neural crest migration in Xenopus by modulating microtubule dynamics. In addition, they show an effect of Gai2 knockdown on Rac1 spatial activation and focal adhesion stability. These are novel discoveries of the study. Some limitations exist in linking Gai2 with downstream effectors that directly or indirectly impact on cytoskeleton and Rac1 small GTPase.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This manuscript examines the role of a G protein, Gai2, in regulating the migration of cranial neural crest cells. Although previous literature has established that heterotrimeric G proteins are involved in cell migration, a central process during embryogenesis and adult homeostasis, the underlying cell biological mechanisms of their activities have not been elucidated. This manuscript rigorously examines the various aspects of Gai2 protein interactions to generate an exciting new paradigm in which Gai2 maintains normal microtubule dynamics by binding to tubulin and EB proteins. This normally dynamic microtubular intracellular environment then promotes cortical actin formation in the leading edge of the migrating cell as well as rapid focal adhesion disassembly by controlling Rac1 activity. Under conditions in which the levels of Gai2 are reduced by MO-mediated knockdown, cells display reduced microtubule dynamics and a decreased catastrophe rate, resulting in slower and more stable microtubules to which EB3 is more persistently associated. A stable microtubule environment leads to enhanced Rac1 activation at the leading edge and stable and larger focal adhesions, resulting in reduced migration. The authors utilize cutting edge approaches to examine the interactions between Gai2 and these other cellular components, taking advantage of the well characterized cell migration model - the cranial neural crest - both in embryos and in cultured explants of these cells.

    Major comments:

    The manuscript is mostly well written (it could use a few minor grammatical corrections), the significance of the problem is well described, and the results are clearly presented with adequate controls. The movies, provided as supplementary material, are of the highest quality and are essential additions to the stills provided in the figures. The data convincingly support the key conclusions of the manuscript.

    Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No

    Would additional experiments be essential to support the claims of the paper? No additional experiments are essential.

    Are the experiments adequately replicated and statistical analysis adequate? The number of embryos/ explants per assay and the number of explant replicates for each assay and the statistical assessments are rigorous.

    Are the data and the methods presented in such a way that they can be reproduced? Mostly, however, the description of the MO used for Gai2 knockdown needs more detail:

    1. Does the MO knockdown both S and L homoeologs of X. laevis? Since the level of GAPDH in Figure 1H also looks reduced in Gai2 MO lane, it should be made clear that the apparent knockdown of Gai2 was normalized to GAPDH, rather than being the results of unequal loading of the gel. Yes, I recognize that Figure 1I says normalized, but this is not stated in the results or the methods. Also, was this experiment done with X. laevis or X. tropicalis? I could imagine that if done in X. laevis, the lack of complete knockdown might be due to only one homoeolog being affected.
    2. The knowledge of the efficacy of knockdown in each Xenopus species provided by the information requested in the previous point, would allow the reader to assess the level of knockdown in the remaining assays. To do this, the authors should tell us which assays were done in which species. I am not suggesting that each experiment needs to be done in each species, only that the information should be provided. If the MO is more effective in X. tropicalis - which assays used this species? If the knock down is partial, as shown in Figure 1H-I, which species this represents in the remaining assays would be useful knowledge.

    Minor comments:

    While prior studies are referenced appropriately, and the text and figures are mostly clear and accurately presented, the following are a few suggestions that would help the authors improve the presentation of their data and conclusions:

    1. The cell biological experiments convincingly demonstrate that knockdown of Gai2 causes cells to move more slowly. It would be a nice addition to bring the explant experimental data back to the embryo by showing whether the slower moving NC cells in morphants eventually populate the BA. DO they cease to migrate or are they just slower getting to their destination? This could be done by performing snail2 ISH at a later stage (34-35?)
    2. There are places in the manuscript where the authors use the terms "silencing" or "suppression" of Gai2, when they really mean reduced translation - their system is not a genetic knockout, as clearly demonstrated in Figure 1H-I. I suggest that more accurate wording be used.
    3. In Figures 1-5 there are scale of bars on the cell images, but these are not defined in any of the figure legends.
    4. The abstract is the weakest section of the manuscript, and would have greater impact if it were more clearly written.

    Referees cross-commenting

    The concerns are fair assessments. However, most can be addressed in the text and by clearer presentation of existing data rather than more experimentation.

    Significance

    The molecular regulation of cell movement is a key feature of a number of developmental and homeostatic processes. While many of the proteins involved have been identified, how they interact to provide motility has not been elucidated in any great detail, particularly in embryo-derived cells (as opposed to cell lines). The results obtained from the presented experiments are novel, in-depth and provide a novel paradigm for how G proteins regulate microtubule dynamics which in turn regulate other components of the cytoskeleton required for cell movement. The results will be applicable to many migrating cell types, not just neural crest cells.

    Because of the application of the data to many types of cells that migrate, the audience is expected to include a broad array of developmental biologists, basic cell biologists and those interested in clinically relevant aberrant cell migrations.

    Reviewer keywords: Xenopus embryology; neural crest gene expression; use of MO-mediated knockdown of gene expression. Not an expert in microtubule dynamics.

  10. Version published to 10.1101/2023.09.07.556733v2 on bioRxiv
  11. Version published to 10.1101/2023.09.07.556733v1 on bioRxiv