Tks5 interactome reveals ER-associated machinery translation in invadosomes
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Abstract
The ability to progress and invade through the extracellular matrix is a characteristic shared by both normal and cancer cells through the formation of structures called invadosomes gathering invadopodia and podosomes. These invadosomes are plastic and dynamic structures that can adopt different organizations depending on the cell types and the environment such as rosettes, dots or linear invadosomes. In this study, we used the specific invadosome marker Tks5 (SH3PXD2A), to identify common features in these different organizations. Tks5 immunoprecipitation coupled with mass spectrometry analysis allowed us to identify common proteins in these different models. We identified elements of the translation machinery, in particular the EIF4B protein, but also endoplasmic reticulum (ER) proteins as part of the invadosome structure. Providing new data on invadosome molecular composition through Tks5 interactome, we identified that ER-associated translation machinery is recruited to invadosome and involved in their formation, persistence and function in all types of invadosomes.
Summary
Invadosomes are invasive F-actin structures exhibiting different organizations that degrade the extracellular matrix. The study uses their universal marker, Tks5, to provide new data about invadosome molecular composition and reveal the role of ER-associated translation machinery in invadosome formation and function.
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Manuscript number: RC-2024-02588
Corresponding author(s): Frederic SALTEL
__1. __Point-by-point description of the revisions
Reviewer #1:
Invadosomes are dynamic, actin-based structures that enable cells to interact with and remodel the extracellular matrix (ECM), playing a crucial role in tumor cell invasion and metastasis. Prior studies by the authors and other groups have established the formation, activation, and appearance of invadosomes. This study demonstrates the following:
Key elements of the translation machinery and endoplasmic reticulum (ER) proteins are constituents of the invadosome structure.
Specific proteins are associated with …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Manuscript number: RC-2024-02588
Corresponding author(s): Frederic SALTEL
__1. __Point-by-point description of the revisions
Reviewer #1:
Invadosomes are dynamic, actin-based structures that enable cells to interact with and remodel the extracellular matrix (ECM), playing a crucial role in tumor cell invasion and metastasis. Prior studies by the authors and other groups have established the formation, activation, and appearance of invadosomes. This study demonstrates the following:
Key elements of the translation machinery and endoplasmic reticulum (ER) proteins are constituents of the invadosome structure.
Specific proteins are associated with distinct invadosome structures.
The researchers utilized two cellular models (NIH3T3-Src and A431 melanoma cell line) and Tks5, a specific invadosome marker, for immunoprecipitation and mass spectrometry, validating the results through fluorescent images, electron microscopy, and time-lapse live imaging.
Major Comments
The manuscript is well-written, with a clear and detailed experimental workflow. Compared to their previous seminal work that first demonstrated invadosomes concentrate mRNA and exhibit translational activity using NIH3T3-Src cells, this study adds details about the specific enrichment of translation proteins for each type of invadosome and the presence of ribosomal and ER proteins. However, the experiments do not further enhance our understanding of the intricate mechanisms linking invadosome structures, function, and translation factors.
Further experiments are needed to better demonstrate the hypothesis of active translation within these structures, including the use of additional cellular models.
To demonstrate the hypothesis of active translation within these structures, we performed the same translation inhibition experiments, using CHX in additional cellular models. Indeed, these experiments were performed on MDA-MB-231 breast cancer cell lines, as well as on Huh6 liver cancer cell lines. Degradation experiments showed the same results as for NIH-3T3-Tks5-GFP and A431-Tks5-GFP, since we were able to observe a significant decrease in the degradation capacities of cells in the absence of translation (see graphs below).
Left: Quantification and representative images of ECM degradation properties of Huh6 cells on gelatin treated (CHX) or not (DMSO) with cycloheximide. Gelatin is stained in green and nuclei in blue. Values represent the mean +/- SEM of n=4 independent experiments (15 images per condition and per replicate) and were analyzed using student t-test.
Right: Left: Quantification and representative images of ECM degradation properties of MDA-MB-231 cells on gelatin treated (CHX) or not (DMSO) with cycloheximide. Gelatin is stained in green and nuclei in blue. Values represent the mean +/- SEM of n=4 independent experiments (15 images per condition and per replicate) and were analyzed using student t-test.
The authors should also investigate the effects of Tks5 silencing on ER-associated translational machinery.
The effects of Tks5 silencing on the ER-associated translation machinery were investigated using a SunSET experiment. We were able to demonstrate that Tks5 silencing had no significant impact on translation in both cellular models since no translation modification was observed between control and siTks5 conditions.
Quantification and relative western blot analysis of the effect of Tks5-targeting siRNA treatment on A431 and NIH-3T3-Src cells by using puromycin quantification. Values represent the mean +/- SEM of n=4 independent experiments and were analyzed using Anova.
How do the authors propose Tks5 is linked to these proteins? Directly or indirectly? Focusing on specific proteins night provide an opportunity to study the molecular mechanisms in greater depth.
Tks5 is a scaffold protein, a multi-domain “bridging molecule” that serve as regulators by simultnneously binding multipe molecular partners. TKs5 contain a PX domain and 5 SAH Domains. Consequently, Tks5 can bind different partners. Moreover, as focal adhesion, invadosome are large macromolecular assemblies. Here, in this study, Tks5 serve as a specific molecular hook, to precipitate partners. At this step, there is no evidence of a direct or indirect link of the translational machineray with Tks5. Even if we can hypothetize un indirect connection. In this version we focused more precisely on a specific and common Tks5 partners, such as EIF4B.
They used chemical inhibitors and siRNA approaches to assess the role of specific players, such as EIF4B, in the proteolytic activity of invadosomes, which can be considered proof of concept. Additional experiments aligning the results with the involved pathways would add molecular details and enhance the manuscript's significance. Resolving these issues is crucial for the manuscript to meet the publication standards for contributing novel and impactful insights to the field.
To better understand the variation of the pathways involved, we first wanted to observe the impact of Eif4b silencing on active translation in both cellular models. To do this, we performed SunSET experiments in both cell models. An experiment was performed for the A431 cell line and the results seem to show little difference between control conditions and conditions in the presence of siEIF4B. Conversely, SunSET experiments in the NIH 3T3 Src cell line show an increase in translation in the presence of siEIF4B.
Quantification of the effect of cycloheximide (CHX) and EIF4B-targeting siRNA (siEIF4B #1 and #2) treatment on A431 and NIH-3T3-Src cells by using puromycin quantification. Values represent the mean +/- SEM of n=1 independent experiment for A431 or n=2 independent experiments for NIH-3T3-Src.
In order to better understand the variation of the signaling pathways involved, spectrometry experiments were performed to compare the variation of the pathways in control conditions and in the presence of siRNA against EIF4B. These results allowed us to provide a better understanding of the variability of the pathways and therefore of the mechanism of action.
Volcano plot of overexpressed and underexpressed proteins after silencing of the EIF4B protein identified by mass spectrometry analysis.
These mass spectrometry experiments allowed us to highlight that the pathway mainly impacted during Eif4b depletion was the Hras pathway. However, this information is given for information purposes only. It would be necessary to look more closely at the Hras pathway to understand what the link with EIF4B and therefore the link with the formation of invadosomes could be.
Table of translation-related proteins or proteins involved in the formation or function of invadosomes that are overexpressed or underexpressed in at least one siRNA of EIF4B.
These experiments also allowed us to highlight that the depletion of EIF4B directly impacts the translation pathway by modulating translation initiation factors as well as ribosomal proteins but also proteins involved in the formation and function of invadosomes such as ADAM17, ACTR5, IGFBP6 RPL22 and RPS6KA5 proteins (see table below). It will be necessary to validate these data and determine their specificity due to the fact that some other proteins appear under-expressed like IGFBP3 and ADAM19. To conclude, to fully understand the exact impact of EIF4B into this process, additional investigations are necessary.
__ __Minor Comments :
A more detailed discussion of the implications of their findings within the broader context of cancer cell signaling and the potential impact on related cancer research areas would further advance our understanding in this area.
This part was added in the new version of the discussion. Indeed, deregulation of the translation is now a hallmark of cancer. This notion is now present in the manuscript and concluded the discussion (see page 12).
Reviewer #1 (Significance (Required)):
General Assessment:
This study offers novel insights into a new function of the invadosome-specific player Tks5 as a molecular crossroad between ER-related translation proteins and invadosomes. The authors suggest that Tks5 could act as a scaffold, supporting the rapid clustering of translation-related proteins during invadosome formation or proteolytic activity. However, a major limitation is the lack of mechanistic exploration. The results do not elucidate how Tks5 mediates the recruitment of these proteins or the specific molecular mechanisms involved.
Advances: The study extends knowledge in the field by confirming the presence of specific markers linked to different invadosome structures and demonstrating the Tks5 interactome's association with translation machinery.
Audience: This study will primarily interest specialists working on invadosomes and, secondarily, those interested in cancer cell signaling, invasion, and metastasis.
Field of Expertise: Invadosome and related signaling pathways in cancer.
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Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary In this work, Normand and her colleagues analyze and compare the interactome of the key invadopodia component, TKS5 (overexpressed as a GFP-tagged protein), in two transformed cell models cultured on different substrates. Potential TKS5 interacting partners are identified including previously known and validated TKS5 interactors, some known to contribute to the mechanism of invadopodia formation and function. Bioinformatic (GSEA) analysis reveals a specific enrichment for proteins related to protein translation and interaction with ER-associated ribosome machinery. Evidence is presented that some of these proteins (RPS6, a component of the 40S ribosomal subunit, and translation factor, EIF4B) localize to TKS5-positive invadopodia in Src-transformed cells. Experiments based on translation inhibitor, cycloheximide, and silencing of EIF4B factor could demonstrate a link between overall protein translation and invadosome formation. Live cell imaging and microscopy analysis of fixed samples could document some proximity between the endoplasmic reticulum network and invadosome rosettes.
Major comments
__ __1- In the Results Section, the IP/proteomics-based pipeline used by Normand and colleagues to identify TKS5 partners is not clearly described and is confusing. Cut-off used to select the proteins in the different classes summarized in Table S1 should be better described. In addition, the nomenclature of the different protein subgroups used in Table S1 is confusing (see minor point#5).
Details have been added in the results section regarding the IP/proteomics section to complete the materials and methods section. As described in the materials and methods section, control versus IP data were quantified by an enrichment ratio ≥ 2. These criteria are the most classically used in the practices analyzed.
For clarity, additional tables have been added for each category (A431/NIH plastic or collagen) and gene names, protein descriptions and abundance ratios have been indicated (Supp table 2, 3, 4 and 5).
2- The effects of cycloheximide treatment or EIF4B silencing on gelatin degradation are clear and convincing. However, these are correlative evidence, and they may reflect a general implication of protein translation in the control of invadopodia function. A direct link between the observed interactions of TKS5 with the protein translation machinery and the formation and/or function of invadopodia is missing.
To demonstrate the direct links between Tks5 and the translation machinery, a fluorophore was used to visualize active translation within invadopodia. We were able to highlight an active translation localized in the rosettes (see figure below). Indeed, we can observe a localized translation within the rosettes. However, these same results were not observed in linear invadosomes where we could not observe any localized translation. We can however hypothesize that it is more difficult to observe a localized translation in linear invadosomes which are much smaller structures than rosettes.
Confocal microscopy images of NIH-3T3-Src cells. The cells were stained for B-actin RNA in green, B-actin in red, nuclei in blue and actin in grey. Scale bar: 20µm, zoom: 5µm.
In order to provide additional elements to show the link between Tks5 and the translation machinery, we performed immunofluorescence experiments by labeling the Sec61 protein. Sec61 is a well-described ER marker that allows the insertion of proteins into the ER but is also a key player in the docking of ribosomes to the ER. We were able to highlight the colocalization between Tks5 and Sec61 in all types of invadosomes, allowing to show the link between the Tks5 protein and the translation machinery. These images were inserted in the manuscript (see Figure 6b).
Confocal microscopy images of NIH-3T3-Src and A431 cells. The cells were seeded on gelatin or type I collagen and stained for Sec61 in red, nuclei in blue and Actin in grey. Scale bar: 20µm, zoom: 5µm.
__ __3- Images showing the interrelations between the ER and the adhesive podosome rosettes are striking (Figure 5). Src-transformed cells forming invadosome rosettes when in contact with the collagen substratum change shape and produce adhesive protrusions towards the substratum. As the ER is a huge compartment that fills the entire cytoplasm, it is maybe not so surprising to observe the ER filling the protrusions and getting close to the rosettes at the tip of these membrane extensions. Again, these observations are essentially correlative and there is no prove of some direct contact between some ER regions and the invadosomes.
For clarity, the contrast of the images has been improved. Thus, time-lapse imaging clearly demonstrate that the ER is not present in all the cytoplasm but is enriched in the destination of the rosettes as well as in the rosettes. Moreover, this is not systematic with all invadosome rosettes (see video 1)
4- Overall, this report is lacking a clear hypothesis or model of what could be the consequence of the interaction of TKS5 and the translation machinery on the formation and/or the activity of the invadosomes in transformed cells.
We performed a sunset experiment to analyze the impact of Tks5 depletion into translation. No variation of global translation was observable in the absence of Tks5 (see results below). Tks5 depletion block invadosome formation. So, the impact on total translation activity cannot be measurable at the cell level, suggesting that invadosome recruit a specific translation machinery. Indeed, even if we obtained a good percentage of Tks5 depletion, around 90%, the impact in total translation activity is not quantifiable. However, we noticed that some specific translation actors are modulated and specifically localized into invadosome structures suggesting that it is more a question of localization and local translation of specific mRNAs, and not a global modification. This is consistent with the fact that Tks5 expression is not altered during tumor cell invasion, and it is just recruited and activated at specific sites to form these invasive structures.
Thus, in this paper, Tks5 only served as an anchor point in order to be able to extract the specific molecular machinery and specific translational actors.
Quantification and relative western blot analysis of the effect of Tks5-targeting siRNA treatment on A431 and NIH-3T3-Src cells by using puromycin quantification. Values represent the mean +/- SEM of n=4 independent experiments and were analyzed using Anova.
Minor comments
1- Discussion Section (page 2). The statement that TKS4 is involved in ECM degradation in podosomes only and not in invadopodia is not correct. TKS4 knock down has been shown to interfere with ECM degradation in Human DLD1 colon cancer cells (Gianni et al. SCIENCESIGNALING Vol 2 Issue 88, 2009) and in in mouse and human melanoma cell lines (Iizuka et al. Oncotarget, Vol. 7, 2016). In addition, an unphosphorylable mutant form of Tks4 blocked invadopodia formation and ECM degradation in Src-transformed DLD1 cells (Gianni et al. Molecular Biology of the Cell Vol. 21, 4287- 4298, 2010). We (this reviewer's team) reported that TKS4 was associated with cortactin-positive invadopodia in MDA-MB-231 and Hs578T triple-negative breast cancer cell lines (Zagryazhskaya-Masson et al. J. Cell Biol. 219, 2020).
The involvement of TKS4 protein in extracellular matrix degradation has been changed in the text (page 2).
2- Discussion Section (page 3). A431 is wrongly referred to as a melanoma cell line; it is a human epidermoid carcinoma cell line.
The text has been modified according to the recommendations, the A431 cell line has been designated as a human epidermoid carcinoma cell line.
3- Results Section (page 4 & 5). The authors compare the proteins they identified as potential TKS5 partners to previously published data by Stilly et al. (based on TKS5 IP like in the present study) and Thuault et al. (TKS5 bioIB). Additionally, authors should mention and discuss previously published data based on TKS5 coIP experiment and Mass Spec analysis similar to the present study, identifying potential TKS5 partners; some of which were similarly found in the present study including proteins involved in translation and ribosome function although these were not the focus of this work (several 40S and 60S ribosomal proteins, see Zagryazhskaya-Masson et al. J. Cell Biol. 219, 2020).
This comparison is now present int the text of the manuscript (page 10).
4- Figure 1b. Matrix degradation is not visible in association with the invadopodia in selected high magnification images in Figure 1a and 1b.
Matrix degradation is indeed not visible in association with invadopodia in the selected high magnification images. Indeed, the imaging techniques used, Interference Refection Microscopy (IRM) do not allow us to observe matrix degradation at the invadosomes, since the reflection also highlights the cells. The aim here was to show only the presence collagen fibers that correspond to inducer of linear invadosome reorganization. It is widely accepted that all these structures are capable of degrading the extracellular matrix.
5- Supplemental table 1. The names of the different lists of proteins in the summary table is not clear and is rather confusing.
For clarity, additional tables have been added for each category (A431/NIH plastic or collagen) and gene names, protein descriptions and abundance ratios have been indicated (Supp table 2, 3, 4 and 5).
6- Supp Figure 1. Please define what is the sample named 'D' (Delta).
The Delta sample corresponds to the material that was not attached to the bead.
7- Results Section (page 5). 'These experiments confirm the correct co-localization between Tks5 and the proteins identified in Tks5 interactome by mass spectrometry analysis.' This statement is too general; in fact, data validate only colocalization between TKS5 and some identified partners, namely CD44 and MAP4.
To be less general, this statement has been modified in the text to show that the data only validate colocalization between TKS5 and certain identified partners, namely CD44 and MAP4.
8- Figure 2e and Figure 3. It would have been nice to show the colocalization of selected proteins and TKS5 in association with collagen fibers to validate that enrichment occurs at matrix/cell contact sites and corresponds to bona fide invadopodia.
As commented above, the reflection highlights the collagen fibers but also the cells. Thus, it is complex in this case to show the colocalization of the selected proteins in association with the collagen fibers with this approach. The other possibility is to stain collagen fibrils, however this kind of approach reduce the quality of interaction between fibers and associated receptors inducing a decrease of linear invadosome formation.
9- Figure 3c (high mag insets). TKS5 and EIF4b do not seem particularly enriched in invadopodia rosettes as compared to the rest of the cytoplasm.
Indeed, we can observe on this image a colocalization of Tks5 and EIF4B in the rosettes without showing an enrichment.
However, the enrichment of EIF4B remains clearly visible in the linear invadosomes and the dots.
10- Figure 4c-f. Treatments (i.e. CHX, siEIF4b) affect gelatin degradation. It would be interesting to assess the capacity of cells to form invadopodia under these conditions.
As demonstrated in this study, the CHX treatment and EIF4B depletion affect the degradation of gelatin. In addition, we were able to show that CHX only impacts the formation of rosettes on gelatin (Figure 4a, 4b and Supp 3).
Moreover, we added in the manuscript the impact of siEIF4B on invadosome formation (Supp Figure 3g). We show that it affects the formation of rosettes as CHX, but also affects the formation of linear invadosomes on collagen by A431 cells.
Quantification of the numbers of invadosomes per cell on gelatin and collagen silencing (siEIF4B) or not (DMSO) for EIF4B in A431-Tks5-GFP and NIH3T3-Src-Tks5-GFP cells. Values represent the mean +/- SEM of n=4 independent experiments (10 images per condition and per replicate) and were analyzed using student t-test.
Reviewer #2 (Significance (Required)):
This study confirms and adds to a previously published report by this research group based on invadosome laser capture microdissection and proteomics revealing that invadosomes contain specific components of the translational machinery, and that protein translation activity is required to maintain invadosome structure and activity (Ezzoukhry et al. Nat Commun 2018). It also adds to a recent study that established a crucial role for ribosome biogenesis in promoting cell invasion in the C. elegans anchor cell invasion model (Development. 2023).
The experimentation presented in this paper is of good quality and convincingly support the authors conclusions of a link between the ER-associated translation machinery and invadosome function in transformed cells. Overall, although this study adds to the emerging idea of an evolutionary-conserved translational control of cell invasion through the extracellular matrix it is mostly correlative and lacking a direct prove that the interaction of TKS5 with components of the translation machinery has a direct contribution to invadopodia function.
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Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary: To invade the surrounding extracellular matrix (ECM), cells organize actin-rich cellular membrane structures capable of ECM degradation, called invadosomes. Depending on the composition and organization of the ECM, cells organize their invadosomes differently. The authors aimed to identify specific and common components of different types of invadosomes: rosettes formed by NIH3T3-Src cells seeded on gelatin, dots formed by A431 cells seeded on gelatin, and linear invadosomes formed by NIH3T3-Src and A431 cells when seeded on fibrillar collagen I. For this, they generated cells stably expressing GFP-Tks5, a ubiquitous constituent of invadosomes, and determined its interactome. They identified 88 common proteins, among which the protein translation machinery was enriched. Whereas general protein inhibition impaired only rosette formation and impaired every type of invadosome-associated degradation, EIF4B inhibition inhibited the formation of every type of invadosomes. They then analyzed the impact of the ER on invadosome formation and degradation activity. First, they documented the presence of the ER in the center of the NIH3T3-Src rosettes and correlated ER presence with rosette initiation and persistence. They then demonstrated that chemical inhibition of Sec61 translocon decreased formation of invadosomes in general.
Major comments:
1- The authors use cells overexpressing GFP-Tks5 for their analysis of Tks5 interactome in the different invadosomes (Fig. 2). The impact of GFP-Tks5 overexpression on invadosome formation and degradation activity should be mentioned.
Depending the cell type the TKS5-GFP overexpression do not increase the number of invadosomes but increase the matrix degradation activity (Di Martino et al 2014); or could impact the number of invadosomes as in B16 cell line (Shinji Iizuka et al, 2016). This point was added in the introduction.
However, the Tks5 overexpression was used fo immunoprecipitation and mass spectrometry analysis. The rest of the study and targets validation are done on wild type cells.
2- Concerning the analysis of the mass spectrometry (MS) data, clarifications would be appreciated:
a. The authors first "determined the specific molecular signature associated with each invadosome organization" (p.4). As I understand it, the proteins in each of these signatures correspond to proteins identified only in a particular type of invadosomes, not in the others. Could the authors indicate the percentage of the total proteins identified for each type of invadosomes that corresponds to the specific molecular signature?
The meaning of the sentence has been changed in the paper to provide more understanding. The term "molecular signature" has been replaced by "specific proteins". Percentages have been added to the tables in Figure 1 Supp.
- __ __ The GSEA pathways related to each of the specific molecular signature were then analyzed and the authors "commonly identified an enrichment in mitochondrial, ER and Golgi proteins" (page 4) (Supp Fig 1c,e,g). Could the authors provide numbers/percentage/statistics? It is not clear to me whether the biological processes (Supp Fig 1b,d,f) are derived from the analysis of the specific molecular signature or of the total proteins identified for each type of invadosomes. Could the authors clarify this point? The percentages of each specific protein category have been added in Figure 1 Supp.
The biological processes (Supp Fig 1b, d, f) arise from the analysis of the molecular signature common to the 4 invadosomes conditions, namely the dots, rosettes and linear invadosomes of A431 and NIH-3T3-Src. Thus, the biological processes arise here from the 88 proteins commonly identified for all types of invadosomes.
The authors also identified "translation proteins" enriched in the specific molecular signature of each type of invadosomes (p.4). They commented on this category, indicating that each type of invadosome contains a specific set of translation-related proteins. This is true, but according to my analysis of the provided tables, the same applies to the other categories as well. Could the authors comment this point? Indeed, some proteins involved in translation can appear specific or common depending the type of invadosome. Our comment is at this step, only suggest that some of this protein should be specific for invadosome and some could be associated to only one organization. Of course, the role of each protein needs to be investigated.
Would similar categories of proteins (translation, ER, Golgi, mitochondrial) appear as enriched if the Tks5 interactome was analyzed as a whole for each type of invadosomes? (the authors may disregard this comment if comment a. is inaccurate). Protein pathways enriched in the different type of invadosome differ, for example, Protein activity GTPase activity, vs cell adhesion molecule binding or hydrolase activity acting on Acid Anyhdrides. This analysis demonstrates and highlights differences between the different invadosome organization. However, we focus on translational proteins, ER proteins for example and calculated the percentage of protein identified and associated with this different structure. We can notice important difference as 3% of translation proteins for rosette vs 9 % for dots in A431 cells. This point suggests that the part of each element can differ.
__ __ The authors identified that "cell adhesion proteins" are specifically enriched in linear invadosomes (page 4) (Supp Fig 1f). This conclusion appears to be based on the analysis of NIH3T3-Src and A431 cells. Could the authors provide more details on how this analysis was performed? Specifically, was the analysis conducted on a mixture of the specific signatures of each of the 2 cell models, or on their shared proteins? Additionally, is this category still enriched if each linear invadosome model is analyzed separately? The analysis was performed on common proteins of linear invadosomes, grouping the two cellular models. The category "cell adhesion protein" is not specifically enriched in linear invadosomes because adhesion proteins are also found in the other groups. However, this category represents a larger percentage in linear invadosomes, thus justifying our choice to highlight it for this category.
__ __ The authors identified 88 proteins common to all types of invadosomes (Fig. 2b) and classified them as validated or not in invadosomes. Could the authors give details on the criteria used for this classification? References for the already validated proteins should also be provided. RTN4 has been described as partially localized at invadopodia formed by MDA-MB-231 cells in Thuault et al., yet the authors classified it as not validated in invadosomes. The RTN4 protein has been moved to the category of proteins identified as localized in at least one invadosomes organization, thank you for this precision.
Please find below the list of papers having among the proteins classification as identified in at least one invadosomes organization, based on literature searches.
ADAM15 : Aspartate β-hydroxylase promotes pancreatic ductal adenocarcinoma metastasis through activation of SRC signaling pathway - Ogawa et al 2019
ADAM19 : The Adaptor Protein Fish Associates with Members of the ADAMs Family and Localizes to Podosomes of Src-transformed Cells - Abram et al 2003
ASPH : Aspartate β-hydroxylase promotes pancreatic ductal adenocarcinoma metastasis through activation of SRC signaling pathway - Ogawa et al, 2019
BAG3 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
CALD1 :
Caldesmon is an integral component of podosomes in smooth muscle cells - Eves et al, 2006
Caldesmon is an integral component of podosomes in smooth muscle cells, Gu et al 2007
Changes in the balance between caldesmon regulated by p21‐activated kinases and the Arp2/3 complex govern podosome formation, Morita et al 2007 CD44 :
The CD44s splice isoform is a central mediator for invadopodia activity, Zhao et al
CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness, Grass et al, 2013
CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts, Chabadel et al, 2007
Macrophages podosomes go 3, Goethem et al 2011 CTTN : ERβ promoted invadopodia formation-mediated non-small cell lung cancer metastasis via the ICAM1/p-Src/p-Cortactin signaling pathway - Wang et al, 2023
EIF4B : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
FNBP1L : Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion and metastasis - Chander et al, 2013
FXR1 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
G3BP1 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
HNRNPA1 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
IGF2BP2 : IMP2 and IMP3 cooperate to promote the metastasis of triple-negative breast cancer through destabilization of progesterone receptor - Kim et al, 2018
ITGA5 : Membrane Proteome Analysis of Glioblastoma Cell Invasion, Mallawaaratchy et al, 2015
LAMP1 : Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts - Chun Tu et al, 2008
MAP4 : A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells, Thuault et al, 2020
MMP14 :
Receptor-type protein tyrosine phosphatase alpha (PTPα) mediates MMP14 localization and facilitates triple-negative breast cancer cell invasion - Decotret 2021
Deciphering the involvement of the Hippo pathway co-regulators, YAP/TAZ in invadopodia formation and matrix degradation - Venghateri 2023 MYH9 :
TRPM7, a novel regulator of actomyosin contractility and cell adhesion 6 Clarck et al, 2006
Bradykinin promotes migration and invasion of hepatocellular carcinoma cells through TRPM7 and MMP2, Chen et al, 2016 NONO : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
NPM1 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
PABPC1 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
PPP1CA : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
PRKAA1 : A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells, Thuault et al, 2020
PTBP1 : The lncRNA MIR99AHG directs alternative splicing of SMARCA1 by PTBP1 to enable invadopodia formation in colorectal cancer cells - Li et al, 2023
RPL10A : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
RPL34 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
RPS4X : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
RRBP1 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
RTN4 : A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells, Thuault et al, 2020
SSB : The PDGFRα-laminin B1-keratin 19 cascade drives tumor progression at the invasive front of human hepatocellular carcinoma - Govaere 2017
STX7 : Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation, Parveen et al, 2022
SYNCRIP : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
THBD : VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis - Cheng-Hsiang Kuo - 2021
YBX3 : Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation, Ezzoukhry et al, 2018
__ __ Page 7, "In addition to translation proteins, the MS analysis highlighted the presence of ER-related proteins such as RTN4, LRRC59 or RRBP1 in all invadosomes linked with Tks5 (Figure 2c)". Is the "ER proteins" category enriched among the 88 common proteins? GSEA analysis on the 88 proteins showed an enrichment in proteins related to ribosomes and mRNA binding.
__ __ The comparative analysis of the TKS5 interactome from NIH3T3-Src-GFP-TKS5 on gelatin (this study) with the proteome of NIH3T3-Src rosettes from Ezzoukhry et al. (Fig 5a and Supp Table 2) should be included in the analysis of the MS data obtained in this study (Fig 2), rather than in the paragraph "Recruitment of ER into invadosome rosettes". Are "ER proteins" enriched? Comparative analysis of the TKS5 interactome of NIH3T3-Src-GFP-TKS5 on gelatin (this study) with the proteome of NIH3T3-Src rosettes from Ezzoukhry et al. was included in Supp Figure 2.
The proteins related to translation are enriched, but not those of the ER.__ __3- Was the localization of the newly identified Tks5 partners, such as RPS6 and EIF4B, but also MAP4 and CD44, to invadosomes analyzed in cells expressing endogenous levels of Tks5? If not, this should be addressed to rule out the possibility that their localization in invadosomes is linked to Tks5 overexpression. Through the figures, it is important to indicate whether cells overexpressing or not Tks5 were used.
The precision on the overexpression of Tks5 has been added in the figures.
The experiments were also carried out on cells not overexpressing Tks5 (see results below). Clarifications have been added in the article to specify that these experiments were carried out on cell lines overexpressing Tks5 but also on WT cell lines not overexpressing Tks5 (data not shown in the paper).
Confocal microscopy images of A431 and NIH-3T36Src cells. The cells were seeded on gelatin or type I collagen and stained for Tks5 in green, actin in red, nuclei in blue and Eif4b in grey. Scale bar: 40µm, zoom: 10µm.
Confocal microscopy images of A431 and NIH-3T3-Src cells. The cells were seeded on gelatin or type I collagen and stained for Tks5 in green, actin in red, nuclei in blue and RPS6 in grey. Scale bar: 40µm, zoom: 10µm.
Confocal microscopy images of A431 and NIH-3T3-Src cells. The cells were seeded on gelatin or type I collagen and stained for Tks5 in green, actin in red, nuclei in blue and MAP4 in grey. Scale bar: 40µm, zoom: 10µm.
4- EIF4B depletion inhibits ECM degradation (Fig 4e-f). The authors should address the impact of EIF4B depletion on invadosome formation. In other words, does EIF4B depletion corroborate the results obtained with CHX treatment, where only rosette formation is inhibited (Fig. 4a and Supp Fig. 3d).
The impact of EIF4B depletion on invadosome formation was studied. We were able to show that EIF4B depletion partly corroborates with the results obtained with CHX treatment, since rosette formation is also inhibited by EIF4B depletion but linear invadosomes formed on collagen by A431 are also inhibited by EIF4B depletion.
These results have been added to the paper (see Figure 3g).
Quantification of the numbers of invadosomes per cell on gelatin and collagen silencing (siEIF4B) or not (DMSO) for EIF4B in A431-Tks5-GFP and NIH3T3-Src-Tks5-GFP cells. Values represent the mean +/- SEM of n=4 independent experiments (10 images per condition and per replicate) and were analyzed using student t-test.
__ __5- The authors treated NIH3T3-Src-KDEL-GFP and LifeAct-Ruby cells with CHX and conclude that "translation inhibition led to the collapse of the rosette structure (Fig 6a, Video 4)" (page 8): could extra time points be added before T300 to appreciate the collapse of actin before the retraction of ER from the center of the rosette. No video 4 is provided. A video 5 is provided but does not correspond to a rosette collapse. The lifetime/dissociation rate of rosettes with and without CHX treatment should be determined.
Live cell imaging has been performed by recording one image every 2 minutes as described in methods. Graphs represent all recorded points along the experiment however we modified scale of original graph included into the manuscript to better appreciate the dissociation of fluorescence intensity curves revealing the collapse of actin before the retractation of ER. We also added a second graph which confirmed our first interpretation.
For video 4, we submitted the videos to make sure there were no errors. So, we can now clearly see the collapse of the rosette in video 4.
Lifeact-mRuby and KDEL-GFP signals were recorded in NIH-3T3-Src cells treated with cycloheximide (CHX; 35µM)
__ __6- Sec61 translocon inhibition by the chemical inhibitor ES1 decreases formation of dots by A431 and rosettes and linear invadosomes by NIH3T3-Src (Fig. 6b). Sec61 siRNA should be analyzed. Does Sec61 localize at invadosomes?
Immunofluorescence on NIH-3T3-Src and A431 WT cell lines were performed and added in the paper showing the localization of Sec61 in invadosomes (Figure 6b). Currently, we did not test siRNA targeting Sec61.
Confocal microscopy images of NIH-3T3-Src and A431 cells. The cells were seeded on gelatin or type I collagen and stained for Sec61 in red, nuclei in blue and Actin in grey. Scale bar: 20µm, zoom: 5µm.
__ __Minor comments:
1- The data of Figure 1 is not totally new, at least plasticity of NIH3T3-Src invadosomes has already been described in Juin A., MBoC, 2012. References to original work should be mentioned.
Indeed, the reference has been added to the text at Figure 1.
2- Page 4 "We realized immunoprecipitation against GFP in both cell lines on plastic and type I collagen conditions": the authors should show/mention that on plastic, cells behave has on gelatin coating.
A sentence has been added to the text to mention this: "Indeed, on plastic, the cells behave as on a gelatin coating and thus form the same types of invadosomes, i.e. dots for A431 cells and rosettes for NIH-3T3-Src cells." (see page 4).
3- The authors compared their MS data to previously published Tks5 interactomes (page 4) (Supp Fig 2a). A study from Zagryakhskaya-Masson et al (PMID: 32673397) identified Tks5 interactome of MDA-MB-231 cells generating linear invadosomes. Could the authors comment this study?
This study shows that FGD1, a guanine nucleotide exchange factor for the Rho-GTPase CDC42 interacts with Tks5 and plays a role in the formation of linear invadosomes. We have added this reference in the manuscript, but we have not found FGD1 in our data. It is possible that the GEF of Cdc42 varies from one cell type to another. This study has been added to the discussion.
4- The comparison of translation proteins found in this study with the ones found in other studies (Supp. Fig. 3 a) should be combined with the paragraph commenting the 88 common proteins (Fig. 2c-d).
For clarity, we decided to separate these two parts. There is indeed a lot of information, so it seemed clearer to us to keep the structure of the figures in this sense.
5- The table Supp Fig 2c listing the proteins present in each of the functional categories enriched among the 88 common Tks5 partners should be included as main figure or a color code representing the different biological processes should be included in Fig 2c.
A color code has been added between the two tables. A sentence has been added in the legends for clarity: "Color codes are according to Table Supp Figure 2c: orange: translation, green: actin cytoskeleton, and blue: adhesion."
__ __6- The SUnSET assay is not correctly untitled and described in the Material and Methods. Indeed, the paragraph refering to it is entitled "Inhibition of translation machinery present in invadosomes" and is a mixture of immunofluorescence and SUnSET protocols.
The SunSET assay materials and methods were modified in the paper in the "Sunset Assay" section as described below:
Sunset assay
Cells were treated with puromycin (10mg/ml) during 10min at 37°C then washed twice in ice-cold PBS for protein extraction as described above in Western Blot section. For negative control we pre-treated cells with the translation inhibitor cycloheximide (35mM) during 10min at 37°C.
7- Figure 4, the decrease in ECM degradation of A431 (GFP-Tks5) cells seeded on gelatin by CHX is not statistically different. The affirmation that "CHX treatment limited degradation activity by A431 and NIH3T3-Src cells on gelatin and collagen matrices" (page 6) should be modulated.
Indeed, thank you for your observation. We realized that incorrect values had been reported. Statistical tests (t-tests) were redone for each CHX condition, and significant results were found for each condition.
8- Page 8, "These results therefore confirm the presence but also the involvement of the ER in the rosette formation and maintenance over time". At this point in the study, there is a correlation between the presence of the ER and rosette persistence but no direct evidence of ER involvement is provided. The authors should moderate their conclusion.
That's absolutely right, the sentence has been modified accordingly (page 8).
9- Fig 5d: the authors should specify in the figure legend what are the red head arrows.
The red arrows show membranes of the endoplasmic reticulum, present at the level of the invadosome rosette. This point was added in the figure legend.
10- Some references are not correct. For example p.10, "MAP4 and LAMP1 were described in podosomes": ref 23 and 26 are studies on invadopodia, not on podosomes.
Corrections have been made to the text, the term podosomes has been replaced by invadopodia (see section references).
11- The authors indicate p.10, "Thanks to mass spectrometry experiments, we were able to show for the first time the presence of translation proteins in linear invadosomes". In their previous study Ezzoukry et al, they showed the localization of overexpressed Caprin1, eEF2 and eEF1A1 translation machinery components in linear invadosomes formed by NIH3T3-Src seeded on fibrillar collagen I. The authors should modulate their affirmations.
Indeed, this sentence has been modulated in the text (see page 10).
12- Could the authors refer to figures in the Discussion.
References to figures were added in the discussion.
Reviewer #3 (Significance (Required)):
This work extends their previous work, Ezzoukhry et al, in which the proteome of rosettes of NIH3T3-Src was identified after laser microdissection. In this work, they had identified protein translation machinery as components of rosettes and its implication in the degradation activity and/or the formation of rosettes and linear invadosomes.
The present study extends the presence of protein translation machinery to other types of invadosomes and the implication of protein translation in invadosome activity and/or formation. It also confirms the presence of ER in the center of rosettes. It suggests that ER-associated translation is required for invadosomes formation and activity. This knowledge will be of interest for the invadosome researcher community.
My expertise is in: cellular biology, invadopodia, ECM degradation, cancer. I do not have sufficient expertise to evaluate the accuracy of the analysis of mass spectrometry data and the quantification of videomicroscopy experiments.
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Referee #3
Evidence, reproducibility and clarity
Summary:
To invade the surrounding extracellular matrix (ECM), cells organize actin-rich cellular membrane structures capable of ECM degradation, called invadosomes. Depending on the composition and organization of the ECM, cells organize their invadosomes differently. The authors aimed to identify specific and common components of different types of invadosomes: rosettes formed by NIH3T3-Src cells seeded on gelatin, dots formed by A431 cells seeded on gelatin, and linear invadosomes formed by NIH3T3-Src and A431 cells when seeded on fibrillar collagen I. For this, they generated cells stably expressing GFP-Tks5, a ubiquitous …
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Referee #3
Evidence, reproducibility and clarity
Summary:
To invade the surrounding extracellular matrix (ECM), cells organize actin-rich cellular membrane structures capable of ECM degradation, called invadosomes. Depending on the composition and organization of the ECM, cells organize their invadosomes differently. The authors aimed to identify specific and common components of different types of invadosomes: rosettes formed by NIH3T3-Src cells seeded on gelatin, dots formed by A431 cells seeded on gelatin, and linear invadosomes formed by NIH3T3-Src and A431 cells when seeded on fibrillar collagen I. For this, they generated cells stably expressing GFP-Tks5, a ubiquitous constituent of invadosomes, and determined its interactome. They identified 88 common proteins, among which the protein translation machinery was enriched. Whereas general protein inhibition impaired only rosette formation and impaired every type of invadosome-associated degradation, EIF4B inhibition inhibited the formation of every type of invadosomes. They then analyzed the impact of the ER on invadosome formation and degradation activity. First, they documented the presence of the ER in the center of the NIH3T3-Src rosettes and correlated ER presence with rosette initiation and persistence. They then demonstrated that chemical inhibition of Sec61 translocon decreased formation of invadosomes in general.
Major comments:
1- The authors use cells overexpressing GFP-Tks5 for their analysis of Tks5 interactome in the different invadosomes (Fig. 2). The impact of GFP-Tks5 overexpression on invadosome formation and degradation activity should be mentioned.
2- Concerning the analysis of the mass spectrometry (MS) data, clarifications would be appreciated:
a. The authors first "determined the specific molecular signature associated with each invadosome organization" (p.4). As I understand it, the proteins in each of these signatures correspond to proteins identified only in a particular type of invadosomes, not in the others. Could the authors indicate the percentage of the total proteins identified for each type of invadosomes that corresponds to the specific molecular signature?
b. The GSEA pathways related to each of the specific molecular signature were then analyzed and the authors "commonly identified an enrichment in mitochondrial, ER and Golgi proteins" (page 4) (Supp Fig 1c,e,g). Could the authors provide numbers/percentage/statistics? It is not clear to me whether the biological processes (Supp Fig 1b,d,f) are derived from the analysis of the specific molecular signature or of the total proteins identified for each type of invadosomes. Could the authors clarify this point?
c. The authors also identified "translation proteins" enriched in the specific molecular signature of each type of invadosomes (p.4). They commented on this category, indicating that each type of invadosome contains a specific set of translation-related proteins. This is true, but according to my analysis of the provided tables, the same applies to the other categories as well. Could the authors comment this point?
d. Would similar categories of proteins (translation, ER, Golgi, mitochondrial) appear as enriched if the Tks5 interactome was analyzed as a whole for each type of invadosomes? (the authors may disregard this comment if comment a. is inaccurate)
e. The authors identified that "cell adhesion proteins" are specifically enriched in linear invadosomes (page 4) (Supp Fig 1f). This conclusion appears to be based on the analysis of NIH3T3-Src and A431 cells. Could the authors provide more details on how this analysis was performed? Specifically, was the analysis conducted on a mixture of the specific signatures of each of the 2 cell models, or on their shared proteins? Additionally, is this category still enriched if each linear invadosome model is analyzed separately?
f. The authors identified 88 proteins common to all types of invadosomes (Fig. 2b) and classified them as validated or not in invadosomes. Could the authors give details on the criteria used for this classification? References for the already validated proteins should also be provided. RTN4 has been described as partially localized at invadopodia formed by MDA-MB-231 cells in Thuault et al., yet the authors classified it as not validated in invadosomes.
g. Page 7, "In addition to translation proteins, the MS analysis highlighted the presence of ER-related proteins such as RTN4, LRRC59 or RRBP1 in all invadosomes linked with Tks5 (Figure 2c)". Is the "ER proteins" category enriched among the 88 common proteins?
h. The comparative analysis of the TKS5 interactome from NIH3T3-Src-GFP-TKS5 on gelatin (this study) with the proteome of NIH3T3-Src rosettes from Ezzoukhry et al. (Fig 5a and Supp Table 2) should be included in the analysis of the MS data obtained in this study (Fig 2), rather than in the paragraph "Recruitment of ER into invadosome rosettes". Are "ER proteins" enriched?
3- Was the localization of the newly identified Tks5 partners, such as RPS6 and EIF4B, but also MAP4 and CD44, to invadosomes analyzed in cells expressing endogenous levels of Tks5? If not, this should be addressed to rule out the possibility that their localization in invadosomes is linked to Tks5 overexpression. Through the figures, it is important to indicate whether cells overexpressing or not Tks5 were used.
4- EIF4B depletion inhibits ECM degradation (Fig 4e-f). The authors should address the impact of EIF4B depletion on invadosome formation. In other words, does EIF4B depletion corroborate the results obtained with CHX treatment, where only rosette formation is inhibited (Fig. 4a and Supp Fig. 3d).
5- The authors treated NIH3T3-Src-KDEL-GFP and LifeAct-Ruby cells with CHX and conclude that "translation inhibition led to the collapse of the rosette structure (Fig 6a, Video 4)" (page 8): could extra time points be added before T300 to appreciate the collapse of actin before the retraction of ER from the center of the rosette. No video 4 is provided. A video 5 is provided but does not correspond to a rosette collapse. The lifetime/dissociation rate of rosettes with and without CHX treatment should be determined.
6- Sec61 translocon inhibition by the chemical inhibitor ES1 decreases formation of dots by A431 and rosettes and linear invadosomes by NIH3T3-Src (Fig. 6b). Sec61 siRNA should be analyzed. Does Sec61 localize at invadosomes?
Minor comments:
1- The data of Figure 1 is not totally new, at least plasticity of NIH3T3-Src invadosomes has already been described in Juin A., MBoC, 2012. References to original work should be mentioned.
2- Page 4 "We realized immunoprecipitation against GFP in both cell lines on plastic and type I collagen conditions": the authors should show/mention that on plastic, cells behave has on gelatin coating.
3- The authors compared their MS data to previously published Tks5 interactomes (page 4) (Supp Fig 2a). A study from Zagryakhskaya-Masson et al (PMID: 32673397) identified Tks5 interactome of MDA-MB-231 cells generating linear invadosomes. Could the authors comment this study?
4- The comparison of translation proteins found in this study with the ones found in other studies (Supp. Fig. 3 a) should be combined with the paragraph commenting the 88 common proteins (Fig. 2c-d).
5- The table Supp Fig 2c listing the proteins present in each of the functional categories enriched among the 88 common Tks5 partners should be included as main figure or a color code representing the different biological processes should be included in Fig 2c.
6- The SUnSET assay is not correctly untitled and described in the Material and Methods. Indeed, the paragraph refering to it is entitled "Inhibition of translation machinery present in invadosomes" and is a mixture of immunofluorescence and SUnSET protocols.
7- Figure 4, the decrease in ECM degradation of A431 (GFP-Tks5) cells seeded on gelatin by CHX is not statistically different. The affirmation that "CHX treatment limited degradation activity by A431 and NIH3T3-Src cells on gelatin and collagen matrices" (page 6) should be modulated.
8- Page 8, "These results therefore confirm the presence but also the involvement of the ER in the rosette formation and maintenance over time". At this point in the study, there is a correlation between the presence of the ER and rosette persistence but no direct evidence of ER involvement is provided. The authors should moderate their conclusion.
9- Fig 5d: the authors should specify in the figure legend what are the red head arrows.
10- Some references are not correct. For example p.10, "MAP4 and LAMP1 were described in podosomes": ref 23 and 26 are studies on invadopodia, not on podosomes.
11- The authors indicate p.10, "Thanks to mass spectrometry experiments, we were able to show for the first time the presence of translation proteins in linear invadosomes". In their previous study Ezzoukry et al, they showed the localization of overexpressed Caprin1, eEF2 and eEF1A1 translation machinery components in linear invadosomes formed by NIH3T3-Src seeded on fibrillar collagen I. The authors should modulate their affirmations.
12- Could the authors refer to figures in the Discussion.
Significance
This work extends their previous work, Ezzoukhry et al, in which the proteome of rosettes of NIH3T3-Src was identified after laser microdissection. In this work, they had identified protein translation machinery as components of rosettes and its implication in the degradation activity and/or the formation of rosettes and linear invadosomes.
The present study extends the presence of protein translation machinery to other types of invadosomes and the implication of protein translation in invadosome activity and/or formation. It also confirms the presence of ER in the center of rosettes. It suggests that ER-associated translation is required for invadosomes formation and activity. This knowledge will be of interest for the invadosome researcher community.
My expertise is in: cellular biology, invadopodia, ECM degradation, cancer. I do not have sufficient expertise to evaluate the accuracy of the analysis of mass spectrometry data and the quantification of videomicroscopy experiments.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this work, Normand and her colleagues analyze and compare the interactome of the key invadopodia component, TKS5 (overexpressed as a GFP-tagged protein), in two transformed cell models cultured on different substrates. Potential TKS5 interacting partners are identified including previously known and validated TKS5 interactors, some known to contribute to the mechanism of invadopodia formation and function. Bioinformatic (GSEA) analysis reveals a specific enrichment for proteins related to protein translation and interaction with ER-associated ribosome machinery. Evidence is presented that some of these proteins (RPS6, a …
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this work, Normand and her colleagues analyze and compare the interactome of the key invadopodia component, TKS5 (overexpressed as a GFP-tagged protein), in two transformed cell models cultured on different substrates. Potential TKS5 interacting partners are identified including previously known and validated TKS5 interactors, some known to contribute to the mechanism of invadopodia formation and function. Bioinformatic (GSEA) analysis reveals a specific enrichment for proteins related to protein translation and interaction with ER-associated ribosome machinery. Evidence is presented that some of these proteins (RPS6, a component of the 40S ribosomal subunit, and translation factor, EIF4B) localize to TKS5-positive invadopodia in Src-transformed cells. Experiments based on translation inhibitor, cycloheximide, and silencing of EIF4B factor could demonstrate a link between overall protein translation and invadosome formation. Live cell imaging and microscopy analysis of fixed samples could document some proximity between the endoplasmic reticulum network and invadosome rosettes.
Major comments:
1- In the Results Section, the IP/proteomics-based pipeline used by Normand and colleagues to identify TKS5 partners is not clearly described and is confusing. Cut-off used to select te proteins in the different classes summarized in Table S1 should be better described. In addition, the nomenclature of the different protein subgroups used in Table S1 is confusing (see minor point#5).
2- The effects of cycloheximide treatment or EIF4B silencing on gelatin degradation are clear and convincing. However, these are correlative evidence, and they may reflect a general implication of protein translation in the control of invadopodia function. A direct link between the observed interactions of TKS5 with the protein translation machinery and the formation and/or function of invadopodia is missing.
3- Images showing the interrelations between the ER and the adhesive podosome rosettes are striking (Figure 5). Src-transformed cells forming invadosome rosettes when in contact with the collagen substratum change shape and produce adhesive protrusions towards the substratum. As the ER is a huge compartment that fills the entire cytoplasm, it is maybe not so surprising to observe the ER filling the protrusions and getting close to the rosettes at the tip of these membrane extensions. Again, these observations are essentially correlative and there is no prove of some direct contact between some ER regions and the invadosomes.
4- Overall, this report is lacking a clear hypothesis or model of what could be the consequence of the interaction of TKS5 and the translation machinery on the formation and/or the activity of the invadosomes in transformed cells.
Minor comments:
1- Discussion Section (page 2). The statement that TKS4 is involved in ECM degradation in podosomes only and not in invadopodia is not correct. TKS4 knock down has been shown to interfere with ECM degradation in Human DLD1 colon cancer cells (Gianni et al. SCIENCESIGNALING Vol 2 Issue 88, 2009) and in in mouse and human melanoma cell lines (Iizuka et al. Oncotarget, Vol. 7, 2016). In addition, an unphosphorylable mutant form of Tks4 blocked invadopodia formation and ECM degradation in Src-transformed DLD1 cells (Gianni et al. Molecular Biology of the Cell Vol. 21, 4287- 4298, 2010). We (this reviewer's team) reported that TKS4 was associated with cortactin-positive invadopodia in MDA-MB-231 and Hs578T triple-negative breast cancer cell lines (Zagryazhskaya-Masson et al. J. Cell Biol. 219, 2020).
2- Discussion Section (page 3). A431 is wrongly referred as to a melanoma cell line; it is a human epidermoid carcinoma cell line.
3- Results Section (page 4 & 5). The authors compare the proteins they identified as potential TKS5 partners to previously published data by Stilly et al. (based on TKS5 IP like in the present study) and Thuault et al. (TKS5 bioIB). Additionally, authors should mention and discuss previously published data based on TKS5 coIP experiment and Mass Spec analysis similar to the present study, identifying potential TKS5 partners; some of which were similarly found in the present study including proteins involved in translation and ribosome function although these were not the focus of this work (several 40S and 60S ribosomal proteins, see Zagryazhskaya-Masson et al. J. Cell Biol. 219, 2020).
4- Figure 1b. Matrix degradation is not visible in association with the invadopodia in selected high magnification images in Figure 1a and 1b.
5- Supplemental table 1. The names of the different lists of proteins in the summary table is not clear and is rather confusing.
6- Supp Figure 1. Please define what is the sample named '' (Delta).
7- Results Section (page 5). 'These experiments confirm the correct co-localization between Tks5 and the proteins identified in Tks5 interactome by mass spectrometry analysis.' This statement is too general; in fact, data validate only colocalization between TKS5 and some identified partners, namely CD44 and MAP4.
8- Figure 2e and Figure 3. It would have been nice to show the colocalization of selected proteins and TKS5 in association with collagen fibers to validate that enrichment occurs at matrix/cell contact sites and corresponds to bona fide invadopodia.
9- Figure 3c (high mag insets). TKS5 and EIF4b do not seem particularly enriched in invadopodia rosettes as compared to the rest of the cytoplasm.
10- Figure 4c-f. Treatments (i.e. CHX, siEIF4b) affect gelatin degradation. It would be interesting to assess the capacity of cells to form invadopodia under these conditions.
Significance
This study confirms and adds to a previously published report by this research group based on invadosome laser capture microdissection and proteomics revealing that invadosomes contain specific components of the translational machinery, and that protein translation activity is required to maintain invadosome structure and activity (Ezzoukhry et al. Nat Commun 2018). It also adds to a recent study that established a crucial role for ribosome biogenesis in promoting cell invasion in the C. elegans anchor cell invasion model (Development. 2023).
The experimentation presented in this paper is of good quality and convincingly support the authors conclusions of a link between the ER-associated translation machinery and invadosome function in transformed cells. Overall, although this study adds to the emerging idea of an evolutionary-conserved translational control of cell invasion through the extracellular matrix it is mostly correlative and lacking a direct prove that the interaction of TKS5 with components of the translation machinery has a direct contribution to invadopodia function.
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Referee #1
Evidence, reproducibility and clarity
Invadosomes are dynamic, actin-based structures that enable cells to interact with and remodel the extracellular matrix, playing a crucial role in tumor cell invasion and metastasis. Prior studies by the authors and other groups have established the formation, activation, and appearance of invadosomes. This study demonstrates the following:
Key elements of the translation machinery and endoplasmic reticulum (ER) proteins are constituents of the invadosome structure.
Specific proteins are associated with distinct invadosome structures. The researchers utilized two cellular models (NIH3T3-Src and A431 melanoma cell line) and Tks5, a …
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Referee #1
Evidence, reproducibility and clarity
Invadosomes are dynamic, actin-based structures that enable cells to interact with and remodel the extracellular matrix, playing a crucial role in tumor cell invasion and metastasis. Prior studies by the authors and other groups have established the formation, activation, and appearance of invadosomes. This study demonstrates the following:
Key elements of the translation machinery and endoplasmic reticulum (ER) proteins are constituents of the invadosome structure.
Specific proteins are associated with distinct invadosome structures. The researchers utilized two cellular models (NIH3T3-Src and A431 melanoma cell line) and Tks5, a specific invadosome marker, for immunoprecipitation and mass spectrometry, validating the results through fluorescent images, electron microscopy, and time-lapse live imaging.
Major Comments:
The manuscript is well-written, with a clear and detailed experimental workflow. Compared to their previous seminal work that first demonstrated invadosomes concentrate mRNA and exhibit translational activity using NIH3T3-Src cells, this study adds details about the specific enrichment of translation proteins for each type of invadosome and the presence of ribosomal and ER proteins. However, the experiments do not further enhance our understanding of the intricate mechanisms linking invadosome structures, function, and translation factors.
Further experiments are needed to better demonstrate the hypothesis of active translation within these structures, including the use of additional cellular models. The authors should also investigate the effects of Tks5 silencing on ER-associated translational machinery.
How do the authors propose Tks5 is linked to these proteins? Directly or indirectly? Focusing on specific proteins might provide an opportunity to study the molecular mechanisms in greater depth.
They used chemical inhibitors and siRNA approaches to assess the role of specific players, such as EIF4B, in the proteolytic activity of invadosomes, which can be considered proof of concept. Additional experiments aligning the results with the involved pathways would add molecular details and enhance the manuscript's significance. Resolving these issues is crucial for the manuscript to meet the publication standards for contributing novel and impactful insights to the field.
Minor Comments:
- A more detailed discussion of the implications of their findings within the broader context of cancer cell signaling and the potential impact on related cancer research areas would further advance our understanding in this area.
Significance
General Assessment:
This study offers novel insights into a new function of the invadosome-specific player Tks5 as a molecular crossroad between ER-related translation proteins and invadosomes. The authors suggest that Tks5 could act as a scaffold, supporting the rapid clustering of translation-related proteins during invadosome formation or proteolytic activity. However, a major limitation is the lack of mechanistic exploration. The results do not elucidate how Tks5 mediates the recruitment of these proteins or the specific molecular mechanisms involved.
Advances:
The study extends knowledge in the field by confirming the presence of specific markers linked to different invadosome structures and demonstrating the Tks5 interactome's association with translation machinery.
Audience:
This study will primarily interest specialists working on invadosomes and, secondarily, those interested in cancer cell signaling, invasion, and metastasis.
Field of Expertise:
Invadosome and related signaling pathways in cancer.
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