Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

  1. Zeinab Rekad
  2. Michaël Ruff
  3. Agata Radwanska
  4. Dominique Grall
  5. Delphine Ciais
  6. Ellen Van Obberghen-Schilling

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Abstract

Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA binding proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically loaded focal adhesions by modulation of integrin signaling and local delivery of β-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks.

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  1. Version published to 10.7554/elife.85165 on eLife
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    Reply to the reviewers

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    The goal of our study was to evaluate the role of the RNA binding protein SAM68 in the regulation of cell adhesion and adaptation of endothelial cells to their extracellular environment. We showed that SAM68 depletion affected endothelial cell behavior by impairing adhesion site maturation and compromising basement membrane assembly.

    We are pleased that the reviewers found our study to be interesting, well written and clear, with findings that are supported by carefully designed experiments. Importantly, we would like to thank the reviewers for their careful analysis of our work and for their clear and constructive comments.

    One common query was whether the regulation of β-actin mRNA localization at adhesion sites, and FN1 gene transcription by SAM68 in endothelial cells involves direct interactions with the mRNA and promoter, respectively. This important point will be addressed with additional experiments in order to strengthen our hypothesis.

    A second point that emerged from the reviews relates to the interdependence of SAM68 multi-layered effects on cell adhesions and FN1 gene transcription. Our response to this issue is discussed below and has been clarified in the revised manuscript.

    Lastly, since in vivo studies are not feasible locally or in a reasonable timeframe, our claim that SAM68 tunes an endothelial morphogenetic program has been toned down in the revised manuscript. Nonetheless, our data clearly show that SAM68 is a major regulator of endothelial adhesion and conditioning of the subendothelial basement membrane.

    Altogether, the proposed experiments and revisions will solidify our data and improve our study thus providing “a significant advance towards understanding the multiple roles of RNA-binding proteins and their coordination in a study system with physiologically relevant connections”, as stated by Reviewer#3.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    To answer critical points raised by the 3 referees, we plan to implement our work with 3 main sets of experiments:

    Set 1 of experiments: Analysis of direct interaction between SAM68 and b-actin mRNA by RIP in endothelial cells according to an improved version of published protocols and results from (Li and Richard, 2016).

    Set 2 of experiments: Analysis of a direct interaction between SAM68 and the FN1 promoter by ChIP in endothelial cells according to published protocols and results from (Li and Richard, 2016).

    Set 3 of experiments: Assessment of the dual functions of SAM68 and their interconnections by i) FN rescue (expression of exogenous FN in SAM68-depleted cells) or ii) by expression of SAM68 mutants.

    In addition, we are generating tools to address the dynamic localization of b-actin in endothelial cells following SAM68 perturbations in endothelial cells (MS2 lentiviral constructs and antisense oligonucleotides designed to abrogate SAM68 recruitment onto b-actin mRNA).

    Below, we describe how these sets of experiment will address Reviewers’ comments and queries in a point-by-point reply.

    Reviewer #1

    • The authors claim that SAM68 interacts with B-actin mRNA to delivery to sites of adhesion only based on siRNA-mediated knockdown experiments. Is the binding of SAM68 to B-actin dynamic process that changes with time? The authors could perform RIP experiments at different stages of cell adhesion - from early points when SAM68 is peripheric to later stages when it homogeneously distributed - to show a potential dynamic interaction with B-actin mRNA.

    β-actin mRNA has been previously identified as a direct target of SAM68 in several published works performed by different groups (Itoh et al., 2002; Klein et al., 2013; Mukherjee et al., 2019). SAM68 binding site has been mapped to a 50 nt length sequence located in the 3’UTR of β-actin mRNA but direct binding of SAM68 onto β-actin mRNA has never been shown in endothelial cells. To this end, we will perform RIP experiments (Set 1 of experiments) to first identify direct recruitment of SAM68 to β-actin mRNA in endothelial cells (as suggested by reviewer 2 as well). Secondly, to address the dynamics of SAM68 interactions with β-actin mRNA we will assess direct interactions of SAM68 with β-actin mRNA at different stages of cell adhesion. These experiments will be conducted using an adapted version of the published SAM68 RIP protocol (Li and Richard, 2016).

    • The article would substantially benefit from live visualisation of B-actin localisation with MS2 tagged transcripts in SAM68 knockdown contexts. This would solidify the proposed mRNA delivery SAM68-mediated mechanism. Although this should not be hard to carry out given the availability of MS2-labelled animals, I understand access to the tools may constitute a major hurdle.

    As mentioned by Reviewer 1, access to MS2-labelled animals and carrying out in vivo experiments in mouse endothelial cells would be a roadblock for our team in the context of this work. Nonetheless, we fully agree that live visualization of β-actin mRNA recruitment at adhesions would solidify our hypothesis. Therefore, we are currently setting up in cellulo experiments in endothelial cells to visualize MS2-β-actin reporters (Yoon et al., 2016), in presence of control or SAM68 binding site-directed antisense blocking oligonucleotides, as previously described (Klein et al., 2013).

    Minor comment:

    • Could the authors run the eGFP-SAM68 movies for longer periods to show the dynamic localisation of the protein during spreading? These experiments would support the data based on fixed material.

    We thank Reviewer 1 for this suggestion and will adjust our imaging pipeline for longer time acquisitions taking caution not to impact cell dynamics due to extended laser exposure.

    Reviewer #2

    • The authors reference previous work defining SAM68 as a beta-actin mRNA interacting protein, however, experiments confirming this in endothelial cells and that this occurs during normal focal adhesion assembly are important.

    This concern will be addressed by Set 1 of experiments (RIP assays) as described in our response to the comments of Reviewer 1.

    • Likewise, experiments addressing how important this action is for focal adhesion function are critical. For example, the beta-actin RNA-binding site of SAM68 could be identified and perturbed to assess the direct impact of this mRNA delivery on FAK-Y397 phosphorylation, focal adhesion assembly, adhesion, cell spreading and migration/sprouting. Without these or similar experiments, the importance of SAM68-mediated beta-actin mRNA delivery is unknown.

    The beta-actin RNA-binding site of SAM68 has previously been identified (Itoh et al., 2002) and antisense blocking oligonucleotides designed to target this sequence have been shown to abrogate SAM68 recruitment onto β-actin mRNA in neurons (Klein et al., 2013). In order to determine whether SAM68 delivery of β-actin mRNA is directly involved in focal adhesion assembly and signalling, we will use the published antisense oligonucleotides to block SAM68 recruitment and assess FAK-Y397 phosphorylation in our bead model.

    • Indeed, if this is not important for FAK-Y397 phosphorylation and focal adhesion assembly, then experiments need to be designed to assess how SAM68 achieves FAK phosphorylation/maturation to provide any significant insight into SAM68 function.

    To address this point, we have generated an RNA binding mutant of SAM68 (KH domain) for analysis of FAK phosphorylation using the bead assay. Importantly, SAM68 is a multi-domain protein that harbors protein/protein interaction domains (SH2 and SH3 binding domains) and it is known to act as a scaffolding protein, in TNFRα signaling for instance (Ramakrishnan and Baltimore, 2011). Therefore, we have also generated lentiviral constructs containing mutations in the SH2 or SH3 binding domains of SAM68 to interrogate its potential signaling adaptor function.

    • The data as presented suggest that a key function of SAM68 is to drive fibronectin (and perhaps other ECM gene) transcription. However, more experiments are needed to validate this conclusion. For example, increased FN1 promoter activity in luciferase assays may be an indirect consequence of feedback to the promoter upon SAM68-mediated action on, amongst other possible actions, focal adhesion signaling, FN transcript splicing or ECM remodeling. Experiments confirming that SAM68 interacts with the endogenous ECM gene promoter would be critical (e.g. via ChIP), as would disruption of the trans-activating action of SAM68 to directly assess the impact of this function (versus modulation of focal adhesion dynamics) on focal adhesion assembly, adhesion, cell spreading and migration/sprouting.

    We fully agree that ChIP experiments to identify recruitment of SAM68 onto the endogenous FN1 promoter in endothelial cells would be required to confirm direct transcriptional activation of the FN1 gene in these cells. Therefore, we will perform these experiments (Set 2) according to a published SAM68 ChIP protocol (to be adapted for endothelial cells) which allowed for the demonstration of specific recruitment of SAM68 onto P21 or PUMA promoters, as well as its transcriptional co-activating activity (Li and Richard, 2016).

    Regarding possible indirect effects of FN1 promoter activity in the luciferase assay shown in Figure 1F on HEK293 cells, we would like to point out that, in addition to their high transfection efficiency, HEK293 cells were chosen for this assay because they display nearly undetectable expression of FN and they are unable to assemble the molecule (even upon overexpression of exogenous FN, see Efthymiou G et al., JCS 2021). Thus, our results using this system support a direct effect of SAM68 on FN promoter activity. This information has been added to the revised text.

    • In parallel, rescue experiments to determine how recovery of endothelial FN expression impacts adhesion, cell spreading, and migration/sprouting (upon SAM68 knockdown) would determine how important this action is to control of endothelial cell behavior.

    Our previous published data showed that autocrine FN expression regulates adhesion, spreading and migration of endothelial cells and that differences in FN expression levels affect assembly of the protein (Cseh et al., 2010; Radwanska et al., 2017). In SAM68-depleted cells, with compromised FN expression, the rescue of FN expression should allow us to uncouple SAM68 functions at adhesion sites from its role as a transcriptional regulator of FN expression (Set 3 of experiments). Expression of exogeneous FN in SAM68-depleted endothelial cells will be performed using lentiviral FN expression constructs described by our team (Efthymiou et al., 2021).

    • Likewise, experiments designed to determine if broader disruption of COL8A1, POSTN, FBLM1 and BGN expression are direct (or indirect, e.g., due to FN disruption) would be important to understand SAM68 function.

    The same set of experiments (Set 3) will be used to analyze by qRT-PCR the expression of COL8A1, POSTN, FBLN1 and BGN mRNAs upon the rescue of FN expression in SAM68-depleted cells.

    • Loss of SAM68 expression in other cell types is known to perturb migration, whereas migration is enhanced in endothelial cells upon SAM68 knockdown. Why would this be the case? Is it that the proposed negative impact of FN production on motility is greater than the positive impact of SAM68 focal adhesion dynamics in endothelial cells versus other cell types? Exploration of the relative impact of these proposed dual functions (using additional experiments as mentioned above) is critical to make sense of these somewhat conflicting observations.

    This point relating to the balance between the negative impact of SAM68-stimulated FN production on motility and the positive impact of SAM68 on focal adhesion dynamics in endothelial cells, is very interesting. Set 3 of experiments, which includes expression of exogenous FN and assessment of cell motility in SAM68-depleted endothelial cells, should allow us to clarify this issue.

    Previous work has implicated phosphorylation of SAM68 as a key trigger of its activity (Locatelli and Lange, 2011, Naro et al., 2022). Additional work exploring the impact of SAM68 phosphorylation on focal adhesion dynamics and ECM gene expression/remodeling (e.g. using phospho-mutants) in this manuscript would have strengthened the message.

    The regulation of SAM68 activity by phosphorylation is a complex question as SAM68 has multiple sites of phosphorylation by serine/threonine and tyrosine kinases. One of these sites (Y440) is a known substrate of Src, a major kinase activated at the cell membrane during adhesion. We are currently generating a Src phosphorylation mutant of SAM68 (Y440F) which could be used to address the impact of SAM68 phosphorylation on integrin signaling and ECM gene expression/remodeling.

    Reviewer #3

    • the authors describe the observed phenotypes as resulting from 'coalescent activities' of SAM68 that play a role in the adaptation of ECs to the extracellular environment. However, it is unclear whether and which of the observed effects result from direct local functions of different SAM68 pools, versus reflecting indirect downstream consequences of one major function. For example, the effects on transcription could be a result of altered adhesion signaling and might occur independently of nuclear SAM68. Or the effects on adhesions could be an indirect consequence of altered transcription of ECM genes, independent of the transient accumulation of SAM68 at the periphery. To support that these are distinct and direct SAM68 functions, the authors would have to provide more evidence for the involvement of SAM68 in the studied processes (e.g. is SAM68 observed by CHIP at promoter regions of ECM genes whose transcription is affected?)

    As recommended by Reviewer 2 as well, we will perform ChIP experiments to document the direct recruitment of SAM68 onto the FN1 promoter (Set 2 of experiments).

    • and try to uncouple them to assess their relative contributions and potential connections in the observed phenotypes (e.g. it would be informative to attempt to rescue the knockdown phenotypes with mutants of SAM68 that cannot be imported into the nucleus or that cannot bind RNA or that cannot be phosphorylated by Src_

    Set 3 of experiments should allow us to uncouple dual functions of SAM68 in endothelial cells. In these experiments, integrin signaling defects will be evaluated in SAM68–depleted cells following the rescue of FN expression. Persistence of the adhesion site defect would indicate that transcriptional activity and adhesion site regulation by SAM68 are distinct events. Moreover, as indicated above, we are generating lentiviral constructs of SAM68 mutants with impaired ability to bind RNA or be phosphorylated by Src (Y440F), in order to assess their effect on integrin signaling.

    Minor comment:

    Also, is the effect of SAM68 depletion on pY397-FAK levels local and/or transient? it would be useful to present data on the total amount of pY397-FAK (by IF or western) in control and si-SAM68 cells at early and late stages of spreading

    This point is very interesting and will be tested at early vs late stage of spreading.

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

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Reviewer #1

    • The authors state that "...both submembranous functions [...] and nuclear functions [...] of SAM68 contribute to the morphogenetic phenotype of angiogenic endothelial cells. Some caution must be taken, as all previous data were obtained from 2D experiments. At this stage it cannot be excluded other mechanisms involved in 3D migration.

    We fully agree with this reviewer’s comment and we have modified the manuscript to take into account the fact that we cannot exclude other mechanisms of action for SAM68 in 3D endothelial cell sprouting experiments However, it is noteworthy that the migration per se of individual cells is not measured in our 3D experiments.

    Minor comments:

    • In Figure 1F, there is a drop-in luciferase activity in cells transfected with higher amounts of vector, rather than an increase with SAM68. Why?

    The luciferase reporter assay is a convenient and well-accepted means of evaluating promoter activities, however, it requires the transfection of increasing amounts of expression plasmids, which often contain strong promoters such as CMV (in our case). Depending on the experimental conditions, a drop in luciferase activity is often observed, due to titration of general transcriptional factors. In our experiments shown in Figure 1F, despite the observed drop in luciferase activity in pcDNA 3.1-transfected cells, transfection of increasing amounts of the SAM68 expression vector induced a significant increase in luciferase activity.

    • The authors claim (and rightly so) that plasmids are hard to transfect into HUVECs when describing luciferase reporter assays. However, they express eGFP-SAM68 (presumably from a plasmid).

    eGFP-SAM68 was delivered and expressed in endothelial cells using a lentiviral vector (this has been specified in the revised manuscript: legend to Movie supplement 1). Although eGFP-SAM68 is successfully expressed, the efficiency of infection is a bit low. Thus, this method is adequate when experiments require observations at the single cell level, such as imaging of endothelial cells expressing eGFP-Sam68. However, the low infection efficiency makes it unsuited for the observation of global effects on the cell population, as is the case for a luciferase assay in which all cells from a given experimental condition are lysed.

    • Some experimental details in the figure legends could be restricted / moved to the methods section.

    • Some typos and British/American spelling inconsistencies (e.g. localisation and localization) need to be corrected throughout the manuscript.

    • Statistical analysis details could be mentioned in figure legends.

    • In page 11, "... proposed to be involved in regulation of early cell adhesion processes and spreading" needs referencing.

    • Y axes in some graphs do not start at 0, which may mislead visual interpretations.

    • "Figure 2-figure supplement 1" in page should read "movie supplement 1"

    We thank Reviewer #1 for these comments which have all been taken into account. Appropriate changes/corrections have been made in the revised version of the manuscript.

    Reviewer #2

    • The title of the manuscript states that SAM68 modulates the morphogenetic program of endothelial cells, yet there are no studies of blood vessel morphogenesis described by this work. Ultimately, in vivo studies of vessel development in SAM68 mutant mice would be required to be able to make this claim.

    We agree that only endothelial cell morphogenesis, and not blood vessel morphogenesis, has been addressed in this study. In light of the reviewer’s recommendation to tone down claims that SAM68 tunes an endothelial morphogenetic program, we have modified the revised manuscript text and title.

    • Place the work in the context of the existing literature (provide references, where appropriate).
      SAM68 has previously been identified as an RNA-binding protein associated with the 'adhesome' that regulates cell motility (Huot et al., 2009a, Locatelli and Lange, 2011, Naro et al., 2022). Here, Rekad and colleagues also probe the action of SAM68 in endothelial cell migration, but find this to be enhanced upon SAM68 knockdown - unlike previous studies demonstrating a reduction in motility in similar experiments in other cell types. Indeed, a detailed discussion of this discrepancy would have been appreciated.

    As recommended by Reviewer #2, we have included a more detailed discussion of this point in the revised manuscript.

    Reviewer #3

    Figure 3C: Is the n=3 indicative that only 3 beads were analyzed? Given the relatively small difference, a larger sample size would be useful.

    We thank the Reviewer for pointing out this mistake. Three independent experiments have been performed with quantification of at least 12 beads for each condition. The manuscript has been corrected accordingly (N=3).

    Page 5-6: The statement 'nearly all adhesion sites in SAM68-depleted cells remained smaller than 0.75 um' doesn't seem to accurately reflect the data presented in the right panel of Figure 1C.

    We have modified the units (µm2) of average adhesion size. Nearly all adhesion sites in SAM68-depleted cells remained smaller than 0.75 µm2

    Page 6: there is a reference to a G418 phosphotyrosine antibody. Do the authors mean 4G10 antibody? Also, there is a mention that materials are listed in Supplemental tables 1 and 2, but these were not attached.

    We thank the Reviewer for having noted these typos, and the fact that we omitted to attached Supplemental Tables 1 and 2. This has been corrected in the revised manuscript, to be submitted with the Supplemental Tables.

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

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Reviewer #1

    • deHoog et al. 2004 (doi.org/10.1016/S0092-8674(04)00456-8) had shown the presence of SAM68 in SICs. Why do the authors believe that the presence of SAM68 in the periphery in endothelial cells does not mark the formation of SICs in these cells?

    Spreading Initiation Centers (SICs) are described as structures involved in the early step of adhesion which contain SAM68, along with other RNA binding proteins (de Hoog et al., 2004) in MRC5 cells. In the same paper, to test whether SICs are a general feature of cell adhesion, authors evaluated the presence of SICS during adhesion of several other cell including endothelial cells (HUVEC). Among the 6 types of cells tested, SICs were not observed in nonfibroblastic cell types. In accordance with this study, we did not observe SICs, as defined by deHoog et al., in endothelial cells plated onto FN

    • In Shestakova et al 2001 (doi.org/10.1073/pnas.121146098), decreased localisation of B-actin mRNA leads to reduced persistence of direction of movement. Was this measured? Is this not seen here because SAM68 is only responsible for B-actin mRNA localisation at early stages of adhesion?

    We thank Reviewer 1 for this comment. After re-analysis of our migration data we did not detect a significant effect on the persistence of migration in our experimental conditions. This could indeed reflect the temporal regulation by SAM68 of b-actin mRNA localization at the leading edge of cells, although we cannot exclude additional defects caused by SAM68 depletion on adhesion stability and lammelipodial protrusion and consequently cell polarity and directional motility.

    • Although the authors claim that altered ECM deposition in SAM68 deficient cells results from altered transcription, they do not address potential misregulation of translation and secretion.

    We did not address misregulated translation here as FN mRNA levels were significantly decreased in SAM68-depleted cells. The decreased transcript levels were accompanied by decreased protein levels. Upon depletion of SAM68, we detected less FN in both “soluble” (conditioned medium) and “insoluble” (ECM-associated) forms, as shown in the western blots of Figure 4-figure supplement 1. We do not believe that SAM68 silencing impacts FN secretion, as we did not observe differential retention of FN in the cytoplasm of SAM68-depleted cells compared to control cells by immunostaining (Figure 4C). Rather, FN staining was strictly fibrillar (ECM-associated) in both control and SAM68-depleted cells, and the intensity profile baseline values were similarly low. This point has been added to the revised manuscript.

    -In, fact the highlight that whilst the level of some mRNAs encoding basement membrane proteins do not decrease in the absence of SAM68, their incorporation was severely affected. This is worth exploring to strengthen the manuscript.

    This issue was not addressed for other basement membrane components. However, the dichotomy in expression and matrix incorporation of certain basement membrane components is most likely due to the sequential and hierarchical nature of ECM assembly. FN is one of the earliest ECM proteins to be assembled and observations from multiple laboratories have shown that FN orchestrates the assembly of multiple matrix components (reviewed in, (Dallas et al., 2006; Marchand et al., 2019)), including COLIV ((Filla et al., 2017; Miller et al., 2014)).

    • Whilst the data presented in figure 7 is convincing, some more detailed mechanistic analyses could help further comprehend 2D and 3D behaviours. Could it be that the nuclear and cellular roles of SAM68 are somewhat decoupled depending on the environment? Could the RNA localisation functions have a critical role in endothelial sprouting and not so much in 2D migration? Some insights are needed to address these questions and wrap up some loose ends. In its current form, this section of the manuscript is too vague.

    It is known that 2D and 3D culture conditions induce differences in cell behavior, notably through differences in the physical (rigid vs pliable) and biochemical (plastic vs fibrin gel) nature of the environments that differentially regulate mechanotransduction, integrin signaling, cell polarity, etc. Here, we show that migration of endothelial cells on rigid 2D substrates is increased upon SAM68 depletion. On the other hand, the ability of cells to align in capillary-like cords and invade a 3D environment is reduced. Mechanistically, effects of SAM68 on FN production and ECM assembly are likely involved in both contexts by providing an adhesive substrate that restricts cell motility in 2D, or bridges neighboring cells and promotes cell survival in 3D. The purpose of performing the sprouting assay presented here in addition to cell migration assays was not to compare the same functions of SAM68 in these 2 different contexts but rather to illustrate that SAM68 controls endothelial cell behavior in both 2D and 3D environments and thus could significantly impact angiogenesis.

    Reviewer#2

    • Many of the findings are rather superficial or observational, and a detailed mechanistic understanding of SAM68 function is lacking. For example, loss of SAM68 expression reduces beta-actin mRNA recruitment to sites of fibronectin-coated bead adhesion, but how is this regulated and what is its impact on focal adhesion dynamics?

    Both the role of beta-actin mRNA localization on cell adhesion dynamics and the impact of reducing this localization have been extensively documented (Katz et al., 2012; Kislauskis et al., 1994; Shestakova et al., 2001), or (Herbert and Costa, 2019) and references therein. In particular, a specific RNA binding protein called ZBP1 has been shown to localize actin mRNA near focal adhesions (Katz et al., 2012) by a Src kinase-dependent mechanism (Hüttelmaier et al., 2005).

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    Dallas SL, Chen Q, Sivakumar P. 2006. Dynamics of Assembly and Reorganization of Extracellular Matrix ProteinsCurrent Topics in Developmental Biology. Academic Press. pp. 1–24. doi:10.1016/S0070-2153(06)75001-3

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    Efthymiou G, Radwanska A, Grapa A-I, Beghelli-de la Forest Divonne S, Grall D, Schaub S, Hattab M, Pisano S, Poet M, Pisani DF, Counillon L, Descombes X, Blanc-Féraud L, Van Obberghen-Schilling E. 2021. Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks. J Cell Sci 134:jcs252957. doi:10.1242/jcs.252957

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  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript Rekad et al. investigate the role of the RNA-binding protein SAM68 in the interactions of endothelial cells with the extracellular matrix. They knockdown SAM68 expression using siRNAs and demonstrate a role in cell migration and angiogenic sprouting. They further extensively characterize the molecular effects of SAM68 loss, including effects associated with adhesions (number and size of focal adhesions, actin cytoskeleton organization, integrin signaling and delivery of beta-actin mRNA to adhesions), as well as effects on transcription and organization of ECM components. They also observe that SAM68 transiently localizes near adhesion sites during early stages of cell spreading, apart from its predominant presence in the nucleus. Based on this, they suggest that SAM68 affects migration and sprouting of endothelial cells through coordinated functions carried out locally both at adhesion sites (where SAM68 controls integrin signaling and mRNA delivery) as well as in the nucleus (where it controls transcription and mRNA splicing). The manuscript is clearly written, and the presented experiments are well-performed. However, the conclusions drawn from these experiments are not fully supported, and in various instances, statements regarding the underlying mechanisms are inferred based on reports of SAM68 functions in other systems.

    For example, the authors describe the observed phenotypes as resulting from 'coalescent activities' of SAM68 that play a role in the adaptation of ECs to the extracellular environment. However, it is unclear whether and which of the observed effects result from direct local functions of different SAM68 pools, versus reflecting indirect downstream consequences of one major function. For example, the effects on transcription could be a result of altered adhesion signaling and might occur independently of nuclear SAM68. Or the effects on adhesions could be an indirect consequence of altered transcription of ECM genes, independent of the transient accumulation of SAM68 at the periphery.

    To support that these are distinct and direct SAM68 functions, the authors would have to provide more evidence for the involvement of SAM68 in the studied processes (e.g. is SAM68 observed by CHIP at promoter regions of ECM genes whose transcription is affected?) and try to uncouple them to assess their relative contributions and potential connections in the observed phenotypes (e.g. it would be informative to attempt to rescue the knockdown phenotypes with mutants of SAM68 that cannot be imported into the nucleus, or that cannot bind RNA, or that cannot be phosphorylated by Src).

    In the absence of additional data, the work is quite descriptive and relies on extrapolations from other studies for supporting the proposed mechanistic model. If further evidence is provided to support it, it would amount to a significant advance towards understanding the multiple roles of RNA-binding proteins and their coordination in a study system with physiologically relevant connections.

    Minor comments for clarifying some existing data:

    Figure 3C: Is the n=3 indicative that only 3 beads were analyzed? Given the relatively small difference, a larger sample size would be useful. Also, is the effect of SAM68 depletion on pY397-FAK levels local and/or transient? it would be useful to present data on the total amount of pY397-FAK (by IF or western) in control and si-SAM68 cells at early and late stages of spreading

    Page 5-6: The statement 'nearly all adhesion sites in SAM68-depleted cells remained smaller than 0.75 um' doesn't seem to accurately reflect the data presented in the right panel of Figure 1C.

    Page 6: there is a reference to a G418 phosphotyrosine antibody. Do the authors mean 4G10 antibody? Also, there is a mention that materials are listed in Supplemental tables 1 and 2, but these were not attached.

    Significance

    See above

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Rekad and colleagues investigate the function of the RNA-binding protein SAM68 in endothelial cell-ECM interactions and remodeling. SAM68 was previously identified as a component of the 'adhesome', and Rekad et. al. further confirms transient co-localization with nascent focal adhesions and reveals that loss of SAM68 triggers disruption to cell spreading and adhesion maturation in endothelial cells. Using fibronectin-coated beads to trigger cell-ECM interactions, Rekad and colleagues further show that pFAK-Y397 is reduced upon SAM68 loss, as is recruitment of beta-actin mRNA to sites of focal adhesion assembly. In parallel, Rekad et. al. uncover additional impacts of SAM68 knockdown on fibronectin deposition, assembly, expression, promoter activity and splicing - as well as broader impacts on expression of other ECM proteins. Overall, this work suggests a dual role for SAM68 in regulation of endothelial focal adhesion dynamics and ECM assembly that negatively regulates cell migration and positively regulates cell sprouting in in vitro assays.

    Major comments:

    • Are the key conclusions convincing?

    The data are well presented and the impact of SAM68 depletion (or over-expression) on focal adhesion state, ECM composition and endothelial cell behavior appear clear. However, the direct function of SAM68 in the observed phenomena remain untested, and interpretation of results either relies heavily on observations based in other systems, or is inadequately followed up with detailed studies of the mechanisms involved. Thus, several conclusions on SAM68 function are not entirely convincing and need to be bolstered with additional experiments.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    Yes, several claims need to be supported with additional experiments (as detailed below). Moreover, the claim that SAM68 tunes an endothelial morphogenetic program is far too speculative based on observations using in vitro assays of vessel branching that do not fully recapitulate vessel morphogenesis. Without additional in vivo work in mouse, or other model systems in which blood vessel morphogenesis can be adequately observed, these claims need to be toned down significantly.

    • Would additional experiments be essential to support the claims of the paper?

    Yes, please see detailed below:

    1. Many of the findings are rather superficial or observational, and a detailed mechanistic understanding of SAM68 function is lacking. For example, loss of SAM68 expression reduces beta-actin mRNA recruitment to sites of fibronectin-coated bead adhesion, but how is this regulated and what is its impact on focal adhesion dynamics? The authors reference previous work defining SAM68 as a beta-actin mRNA interacting protein, however, experiments confirming this in endothelial cells and that this occurs during normal focal adhesion assembly are important. Likewise, experiments addressing how important this action is for focal adhesion function are critical. For example, the beta-actin RNA-binding site of SAM68 could be identified and perturbed to assess the direct impact of this mRNA delivery on FAK-Y397 phosphorylation, focal adhesion assembly, adhesion, cell spreading and migration/sprouting. Without these or similar experiments, the importance of SAM68-mediated beta-actin mRNA delivery is unknown. Indeed, if this is not important for FAK-Y397 phosphorylation and focal adhesion assembly, then experiments need to be designed to assess how SAM68 achieves FAK phosphorylation/maturation to provide any significant insight into SAM68 function.
    2. The data as presented suggest that a key function of SAM68 is to drive fibronectin (and perhaps other ECM gene) transcription. However, more experiments are needed to validate this conclusion. For example, increased FN1 promoter activity in luciferase assays may be an indirect consequence of feedback to the promoter upon SAM68-mediated action on, amongst other possible actions, focal adhesion signaling, FN transcript splicing or ECM remodeling. Experiments confirming that SAM68 interacts with the endogenous ECM gene promoter would be critical (e.g. via ChIP), as would disruption of the trans-activating action of SAM68 to directly assess the impact of this function (versus modulation of focal adhesion dynamics) on focal adhesion assembly, adhesion, cell spreading and migration/sprouting. In parallel, rescue experiments to determine how recovery of endothelial FN expression impacts adhesion, cell spreading and migration/sprouting (upon SAM68 knockdown) would determine how important this action is to control of endothelial cell behavior. Likewise, experiments designed to determine if broader disruption of COL8A1, POSTN, FBLM1 and BGN expression are direct (or indirect, e.g. due to FN disruption) would be important to understand SAM68 function.
    3. The title of the manuscript states that SAM68 modulates the morphogenetic program of endothelial cells, yet there are no studies of blood vessel morphogenesis described by this work. Ultimately, in vivo studies of vessel development in SAM68 mutant mice would be required to be able to make this claim.
    4. Loss of SAM68 expression in other cell types is know to perturb migration, whereas migration is enhanced in endothelial cells upon SAM68 knockdown. Why would this be the case? Is it that the proposed negative impact of FN production on motility is greater than the positive impact of SAM68 focal adhesion dynamics in endothelial cells versus other cell types? Exploration of the relative impact of these proposed dual functions (using additional experiments as mentioned above) is critical to make sense of these somewhat conflicting observations.
    • Are the suggested experiments realistic in terms of time and resources?

    Yes, although this will depend on local availability of personnel and resources.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes

    Minor comments:

    • Specific experimental issues that are easily addressable.

    n/a

    • Are prior studies referenced appropriately?

    Yes

    • Are the text and figures clear and accurate?

    Yes

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    n/a

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    Here, Rekad and colleagues identify SAM68 as a potential key modulator of focal adhesion dynamics, as its knockdown impacts focal adhesion maturation, signaling and delivery of beta-actin mRNA. Moreover, the authors identify SAM68 expression as being critical for correct ECM gene expression and assembly. Finally, Rekad show that loss of SAM68 expression impacts endothelial cell migration and sprouting in in vitro assays. Overall, these observations identify SAM68 as a key regulator of endothelial cell-ECM interactions that could play an important role in regulating endothelial cell morphogenesis in vivo. However, the mechanistic basis of SAM68 function in focal adhesion dynamics and ECM remodeling still remain unclear. Additionally, if these activities reflect direct actions of SAM68 on focal adhesions and/or ECM expression/remodeling or are indirect consequences of one effect on the other remains unclear. Finally, relevance to blood vessel morphogenesis in vivo also remains unclear.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    SAM68 has previously been identified as an RNA-binding protein associated with the 'adhesome' that regulates cell motility (Huot et al., 2009a, Locatelli and Lange, 2011, Naro et al., 2022). Here, Rekad and colleagues also probe the action of SAM68 in endothelial cell migration, but find this to be enhanced upon SAM68 knockdown - unlike previous studies demonstrating a reduction in motility in similar experiments in other cell types. Indeed, a detailed discussion of this discrepancy would have been appreciated. However, the work by Rekad et. al. goes further than previous studies to convincingly demonstrate that SAM68 expression impacts cell-ECM interactions - although the mechanisms of this action are les clear. Previous work has implicated phosphorylation of SAM68 as a key trigger of its activity (Locatelli and Lange, 2011, Naro et al., 2022). Additional work exploring the impact of SAM68 phosphorylation on focal adhesion dynamics and ECM gene expression/remodeling (e.g. using phospho-mutants) in this manuscript would have strengthened the message.

    • State what audience might be interested in and influenced by the reported findings.

    The work would be of interest to audiences studying the molecular basis of cell-ECM interactions and/or the broader vascular biology field.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Keywords: Vascular biology. Endothelial. Vascular development.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This interesting manuscript by Rekad et al. explores unappreciated roles of SAM68 in the context of endothelial biology and angiogenesis. The authors apply a series of in vitro assays to demonstrate cytoplasmic and nuclear functions of SAM68 during endothelial cell adhesion, remodelling and ECM deposition. In accordance with other studies, SAM68 localises to the cell periphery during early stages of adhesion, and it is involved in integrin activity. In its absence, FAK signalling is impaired and focal adhesions fail to mature. The involvement of SAM68 in adhesion goes hand-in-hand with its RNA localisation roles during the distribution of B-actin transcripts towards sites of adhesion. On the other hand, SAM68 induces FN deposition, presumably via the positive regulation of FN1 transcription. The abundance of cellular isoforms of FN1 mRNAs are particularly reduced in the absence, suggesting a role in alternative splicing. The authors claim that this is achieved through transcription regulation rather than direct modulation of splicing factors. Other transcripts downstream of SAM68 include basement membrane components, suggesting that its nuclear activity serves as another level of control of adhesion. Finally, the authors claim that whilst the loss of SAM68 enhances motility in 2D, presumably due to the reduced ECM deposition, it reduces endothelial cell invasion in 3D models of angiogenesis.

    Major comments:

    Overall, the article is well written, clear and the findings are supported by carefully designed experiments. The statistical methods seem adequate, and the information provided is likely to allow reproducibility. However, some of results are rather preliminary and could be supported by further experimental work:

    • deHoog et al. 2004 (doi.org/10.1016/S0092-8674(04)00456-8) had shown the presence of SAM68 in SICs. Why do the authors believe that the presence of SAM68 in the periphery in endothelial cells does not mark the formation of SICs n these cells?
    • The authors claim that SAM68 interacts with B-actin mRNA to delivery to sites of adhesion only based on siRNA-mediated knockdown experiments. Is the binding of SAM68 to B-actin dynamic process that changes with time? The authors could perform RIP experiments at different stages of cell adhesion - from early points when SAM68 is peripheric to later stages when it homogeneously distributed - to show a potential dynamic interaction with B-actin mRNA.
    • The article would substantially benefit from live visualisation of B-actin localisation with MS2 tagged transcripts in SAM68 knockdown contexts. This would solidify the proposed mRNA delivery SAM68-mediated mechanism. Although this should not be hard to carry out given the availability of MS2-labelled animals, I understand access to the tools may constitute a major hurdle.
    • In Shestakova et al 2001 (doi.org/10.1073/pnas.121146098), decreased localisation of B-actin mRNA leads to reduced persistence of direction of movement. Was this measured? Is this not seen here because SAM68 is only responsible for B-actin mRNA localisation at early stages of adhesion?
    • Although the authors claim that altered ECM deposition in SAM68 deficient cells results from altered transcription, they do not address potential misregulation of translation and secretion. In, fact the highlight that whilst the level of some mRNAs encoding basement membrane proteins do not decrease in the absence of SAM68, their incorporation was severely affected. This is worth exploring to strengthen the manuscript.
    • Whilst the data presented in figure 7 is convincing, some more detailed mechanistic analyses could help further comprehend 2D and 3D behaviours. Could it be that the nuclear and cellular roles of SAM68 are somewhat decoupled depending on the environment? Could the RNA localisation functions have a critical role in endothelial sprouting and not so much in 2D migration? Some insights are needed to address these questions and wrap up some loose ends. In its current form, this section of the manuscript is too vague.
    • The authors state that "...both submembranous functions [...] and nuclear functions [...] of SAM68 contribute to the morphogenetic phenotype of angiogenic endothelial cells. Some caution must be taken, as all previous data were obtained from 2D experiments. At this stage it cannot be excluded other mechanisms involved in 3D migration.

    Minor comments:

    • "Figure 2-figure supplement 1" in page should read "movie supplement 1"
    • Could the authors run the eGFP-SAM68 movies for longer periods to show the dynamic localisation of the protein during spreading? These experiments would support the data based on fixed material.
    • In Figure 1F, there is a drop in luciferase activity in cells transfected with higher amounts of vector, rather than an increase with SAM68. Why?
    • The authors claim (and rightly so) that plasmids are hard to transfect into HUVECs when describing luciferase reporter assays. However, they express eGFP-SAM68 (presumably from a plasmid).
    • Some experimental details in the figure legends could be restricted / moved to the methods section.
    • Some typos and British/American spelling inconsistencies (e.g. localisation and localization) need to be corrected throughout the manuscript.
    • Statistical analysis details could be mentioned in figure legends.
    • Y axes in some graphs do not start at 0, which may mislead visual interpretations.
    • In page 11, "... proposed to be involved in regulation of early cell adhesion processes and spreading" needs referencing.

    Significance

    This is a very interesting study that details the multi-layered activity of the RBP SAM68. Although many of the individual roles had been unveiled in other cell types, here the authors suggest that this protein simultaneously orchestrates several aspects of endothelial cell adhesion via distinct routes. Thus, the study may be most relevant for researchers working in the fields of developmental and pathological angiogenesis.

    However, the work falls short from being a conceptual advance in its current form, as some conclusions are not fully backed by experimental evidence.

    My expertise: RNA localisation

  6. Version published to 10.1101/2022.11.16.516790v1 on bioRxiv