Plakins are involved in the maintenance of epithelial polarity

  1. Juliana Geay
  2. Yoran Margaron
  3. David Gentien
  4. Fabien Reyal
  5. Alain Puisieux
  6. Laurent Blanchoin
  7. Laurent Guyon
  8. Manuel Théry

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Abstract

As epithelia are the interface between the organism and the external environment, they are often subject to damage and must be frequently renewed. However, maintaining epithelial integrity during this renewal is challenging, and loss of cell polarity is a potent inducer of tumorigenesis. In this study, we used transcriptomic data from breast cancer cells at different stages of tumor development to identify molecular changes associated with the early stages of tumor transformation. We correlated these protein expression profiles with either cell polarity defects or cell progression along the epithelial-to-mesenchymal transition (EMT). We identified plakins, namely epiplakin (EPPK1), desmoplakin (DSP) and periplakin (PPL), that were downregulated in cells that had lost their epithelial polarity and also downregulated in cells that had progressed through EMT. We further tested them experimentally by knocking down their expression in a non-tumorigenic epithelial breast cell line (MCF10A). We demonstrated their causal role in the loss of polarity, as revealed by the misorientation of the nuclear centrosome vector. We also found that vimentin, a marker of EMT, was overexpressed in plakin knocked-down cells, suggesting that plakins may have both a structural and a regulatory role in maintaining the epithelial state.

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    Reply to the reviewers

    Response to the three reviewers:

    We thank the reviewers for the time they spent reading and evaluating our work, and for their comments and constructive criticisms.

    The three reviewers contested the novelty and significance of our findings. Their main arguments were that the role of plakin/desmosomes in the regulation of epithelial polarity is already known and that our work does not provide any novel mechanistic link between them and the process of cell polarization.

    To the first point we would like to argue that although a general relationship between plakins and cytoskeletal filaments networks, and notably cytokeratin, has been involved in the regulation of both intercellular junction strength and cell migration, their involvement in the asymmetric positioning of organelles in polarized cells has not yet been proposed nor demonstrated. However, in the light of reviewers’ comments, we admit that our wording has been misleading and that we have used the term “epithelial polarity” when it would have been more rigorous to use the term “asymmetric centrosome position in polarized epithelial cells” to describe our observations. We have modified our text to make this clearer and to streamline our descriptions and conclusions on the regulation of centrosome position and the associated asymmetry of the microtubule network. Considering this, we would like to stress out that our discovery about the specific involvement of three plakins (epiplakin, periplakin and desmoplakin) in the regulation of centrosome position in epithelial cells is novel (see our more detailed argumentation below) and fully demonstrated with our data. We insist that these discoveries are significant since we identified these plakins thanks to the changes of their expression levels in two set of cell lines representing progressive stages of mammary breast cancer. Finally, it is important to stress out that our experimental approach is also original since we used a cellular metric, the centrosome position, to interpret and sort transcriptomic data sets. This strategy of mixing cell biology and bioinformatics has proved fruitful and is thus likely to also become influential.

            To support our argumentation that the identification of the role of plakins in the regulation of epithelial cell polarity is novel, we searched for the words “polarity” and “(epi/peri/desmo)plakin” in PubMed.
    • “Polarity and epiplakin” returned 1 review (PMID 24352042)
    • PMID 24352042: It is a review that we cited, in which it is argued that plakins contribute to cell polarity as they bind to all cytoskeleton filaments and connect them with intercellular junctions. The section dedicated to polarity referred to two specific studies: one about BPAG1e, a member of the plectin family of plakin which is involved in the front-rear polarity of migrating keratinocytes, and one about the spetraplakin MACF1, which crosslinks actin and microtubules and is involved in the polarization of epidermal stem cells. The review also refered to the role of plectin in the regulation of centrosome position by attaching it to intermediate filaments.
    • “Polarity and periplakin” returned 1 review, the same as above, and 2 experimental papers (PMID 23777851 and 18823282)
    • PMID 23777851: It is a study on the protein expression profiles of skin cells derived from patients with Atopic dermatis. Authors found that TH17 cytokines, a inflamatory pathway involved in the differentiation and polarization of naive lymphocytes, was activated and the expression of TH17-related molecules was negatively correlated with periplakin.
    • PMID 18823282: It is a characterization of the ubinuclein, which is known to be essentially nuclear but was could be localized to lateral cell borders in differentiated keratinocytes characterized by the expression of involucrin and periplakin.
    • “Polarity and desmoplakin” returned 87 references, most of them related to the role of desmosomes in the establishment of the apical pole of epithelial cells, but only two of those references are specifically related to the centrosome. They showed that CSSP1 and ninein, two centrosomal proteins, can bind to desmosomes via desmoplakin (PMID 26241740, 17227889). But they are not related to centrosome positioning. The two papers are now cited in our discussion anyway. Based on this search, it seems to us that the establishment of the causal role of these three plakins in the role of centrosome position in polarized epithelial cells is clearly novel.

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

    Summary The manuscript by Geay and colleagues examine potential regulators of centrosome positioning in an immortalised breast cell line in vitro on micropatterns that promote cell doublet formation. The authors mine expression data from breast cancer cell lines in vitro to identify microtubule-related transcripts that are potentially downregulated in cells with a mesenchymal phenotype. The authors identify some Plakin proteins, which upon depletion, are reported to change centrosome positioning relative to junctions. The authors propose that plakins are involved in the maintenance of epithelial polarity.

    Major comments I applaud the authors for attempting to identify novel regulation of epithelial polarity. However, I am sorry to say that this manuscript is overtly preliminary. It is a collection of observations without any mechanistic insight (described below). Despite what I write below, I apologise in that these shortcomings as so extensive that I cannot recommend experiments that would 'fix holes', without essentially writing an entirely new project. Even after addressing the points below, I think it unlikely that the observations would make a coherent, mechanistic contribution to the field of epithelial polarity. I do not like to give reviews like this, but unfortunately, the submission of such preliminary works puts us in this position.*

    Authors: It is correct that we did not investigate the underlying mechanism, and thus our work is preliminary from this point of view, but we provided the first set of evidence that the three plakins (epiplakins, periplakins and desmoplakins) are involved in the regulation of centrosome position and the associated asymmetry of the microtubule network in polarized epithelial cells. This identification was far from obvious, and relied on an unusual way to exploit transcriptomic. Data, which we think is quite valuable. We correlated the level of transcripts to a quantitative measurement of cell organisation (the distribution of nucleus-centrosome vectors). This strategy is novel and proved useful since we identified novel regulators of centrosome positioning.

    • 'Epithelial polarity' Throughout manuscript the authors refer to a 'polarity score' and the term 'epithelial polarity' when what they have actually measured is a specific angle of orientation of centrosomes in cell doublets in vitro. This is an overstatement and adds confusion. The term 'epithelial polarity' has overtones of a polarised epithelium, which such doublets do not model. There is no mechanistic investigation into how this polarity score relates to the ability to form a polarised epithelial monolayer, with apical-basal polarity orientations, either a monolayer on a substrate or a monolayer surrounding a single central lumen, such as these MCF10A cells are often used for in 3-dimensional culture. I suggest that the authors simply mention what they actually measure (and in their own words): "coordination of the centrosome along the nucleus-junction axis." *

    Authors: This is correct and we apologize for the confusion. We have now corrected the text and refer specifically to the “position of the centrosome in polarized epithelial cells” instead of “epithelial polarity”. However, it should be noted that we and others already showed that this position is relevant to the establishment of polarity in vitro in 3D culture, and in vivo in developing mouse embryo (Rodriguez-Fraticeeli et al., J Cell Biol, 2012) (Burute et al., Dev Cell, 2017). We have now added a paragraph at the end of the introduction to clarify this point and justify our experimental approach.

    • In Figure 1A-C, cell doublets are reported and apparently quantified to measure a 'polarity score', which is the angle of orientation of centrosomes in cell doublets. Yet, there is no clear information that explains how the cutoff for what defines this polarity score is generated (e.g. why is the cutoff point chosen to be where it is?), or what it means for epithelial polarity (e.g. why is this cutoff point important to be at that site?). Moreover, there is no indication that these cells actually form connected doublets. Labelling and quantitation of potentially connected cells is absent. Do these actually form junctions to the same extent, such that any differences have been exhaustively excluded to be only from the centrosome orientation, rather than cell spreading and cell-cell contact differences (that would alter geometry)? In addition, statistical analysis for part C is missing. *

    Authors: First, it is true that the geometrical sectioning of cells in order to define a region where centrosomes are considered as polarized toward the junction is arbitrary. But isn’t it the case for most thresholds in image analysis? This is how it has been done in all studies of the polarity of migrating cells during would healing for example. What we think is key here, is that the chosen angular sector for polarized centrosomes, is the same for all conditions, so it allowed us to compare the frequency of polarized centrosome based on this criterium.

    Second, it is also true that for the sake of conciseness we did not show too many data about the characterization of the doublets in order to focus on the criteria that we used for our study. But we analyzed the shape of the doublets. In this example below, we measured how “pinched” were the doublet as compared to a a fully convex envelope. Small intercellular junctions lead to high difference between the area of the convex hull and the area of the doublets. However, we did not find that doublets of comparable cell lines with distinct polarity index, such as HCC1937 and HCC1143, had distinct junction length:

    Finally, there is no statistical analysis for in the histogram shown in Figure 1C since we did not compare the polarity index of the different cell lines. We related them to their transcriptomic profiles (Figure 1D).

    3.

    *Fig 1D, 2A,B present select example genes correlated with either polarity score or EMT score (Fig 1D, 2B). It is unclear what insight providing select genes from many that are changed provides. In Fig 2A, an apparent EMT score (seemingly derived from mining of existing expression data not from this laboratory) is provided, ranked by an EMT. No description is provided for what these alterations are (e.g. what is a 'HME_Ras_Twist1E12_TGFb' sample?). Further, what this is supposed to indicate as a mechanistic insight is unclear. *

    Authors: These panels illustrate examples of protein for which the level of transcripts was well correlated (negatively or positively) to the polarity or EMT scores of the various cell lines we tested. We did not describe again these cell lines and referred to the study where they have been described in details since the conditions leading to their phenotypes were less relevant than the consequence on gene expression and EMT progression, which were described in our text and data. There was no specific value in the chosen proteins in Figure 1D and 2B, they simply illustrate how various transcript levels can be compared, and potentially correlated, to geometrical measurement (in the case of the polarity score, 1D) or to a identity measurement (in the case of the EMT score, 2B). There is no mechanistic insight at this stage. Figure 1 and 2 only illustrate the novel method we proposed to extract information about cell architecture or cell identity from transcriptional data sets. The most valuable information will come in Figure 3 in which we will cross these two pieces of information.

    • Figure 3 is highly preliminary. The entirety of Figure 3 is a correlation plot between EMT score and polarity score for microtubule-related transcripts. *

    Authors: We respectfully disagree. Some misunderstanding might explain reviewers’ comment. This is not a “correlation plot between EMT score and polarity score for microtubule-related transcripts”. The values are not the scores but the correlation between the transcript level and the scores (which were described in Figure 1D and 2B that we discussed in the two previous comments of the reviewer). This is much more informative and definitely not preliminary, since it revealed potential structural (polarity score) and functional (EMT score) implications of proteins that were not known before. Proteins on the top-left or bottom right of the graph are all candidate to influence EMT by acting on the structural polarisation of cells.

    *The authors state: "The graph showed an overall negative trend, which means that many genes were positively correlated with an EMT in HME were instead negatively correlated with epithelial polarity of TNBCs (Figure 3). This was expected and confirmed that the progression along EMT is associated with a loss of epithelial polarity." No statistical analysis is presented, no correlation scores and indication of robustness is provided. It is unclear how this provides any mechanistic insight. The authors themselves state that this association is expected. *

    Authors: We apologize for this. We now reported the statistical analysis that confirmed our previous description of a negative trend in this graph. Pearson correlation coefficient is -0.35 (p-value = 0.00023, 95% confidence interval: [-0.50, -0.17]. We have added these details in the main text and Material and Methods. It is correct that this tendency could be expected by considering various studies together, but it is still better when rigorously demonstrated.

    *Moreover, the authors state "Interestingly, three plakins, namely epiplakin (EPPK1), desmoplakin (DSP) and periplakin (PPL) all appeared as clear outliers (Figure 3)." How is an outlier defined & why is this clear? Is the association of these key cell-adhesion molecules with an epithelial cell state novel or known? *

    Authors: We define classically outliers as genes with a score higher than the 75th Percentile + 1.5 times the InterQuartile Range (IQR). 7 genes were outliers, including the three plakins. We have now detailed the procedure in a dedicated section in Material and Methods.

    • Figure 4. The authors perform siRNA-mediated depletion of Desmoplakin, Epiplakin and Periplakin in MCF10A cells. The authors report, "Interestingly, knocked-down cells in culture displayed abnormal shapes, being more elongated and less cohesive (Figure 4C)." No quantitation of such changes are provided. Moreover, cells with KD appeared to be at lower density. Can the authors exclude that these are not merely density-dependent effects.*

    Authors: This is really just a description of the images. The densities didn't seem that different to us, but it is true that we can't rule out a density effect. We didn't do a detailed quantitative description of these phenotypes because they were not central to the argument about centrosome position. However, we thought these images of knock-down cells were worth showing.

    • Throughout the work, the polarity index is reported from plakin depletion conditions with data from a reported 3 independent experiments seemingly pooled (no indication of graph of which independent experiment each data point comes from). Is the statistical analysis performed (missing in Fig 4E, present in Fig 5A-C, S2, S3) from pooled data? If so, this is in appropriate and should be from the averages of independent experiments, to understand batch effects. If not from pooled data, please alter graphs to display this appropriately. *

    Authors: We showed only one data set per conditions to avoid graph over-crowding. We know show these 3 different experiments with distinct colors in the graphs (SuperPlots). Noteworthy, the exact same experiments were performed with another set of siRNA for each of the three plakins and they show exactly the same effect (see Figure S2).

    • Figure 5A. It is unclear how F-actin is measure in the images. Is F-actin labelling a truly representative proxy for junction length? *

    Authors: This is correct, we assumed that the frontier between the two cells, which could be seen with F-actin, corresponded to the intercellular junction.

    • Fig 5C. Why are images of vimentin now provided not on micropatterns? The labelling of vimentin in siPeriplakin cells does not look appropriately controlled for by the other cell conditions. siPeriplakin is clearly at the edge of a colon, whereas this is not clear whether an appropriate region is labelled in the other conditions. *

    Authors: We performed experiments on micropattern when the aim was to characterize the localization of a protein or a compartment, since micropattern normalize cell shape and orient cell architecture. Here our aim was to visualize the global amount of a protein, so micropatterns were not needed. Actually, western blots would have been better suited for these experiments. But these were the data we had. Pictures that were shown have all been taken at the edge of a colony. The boundary is visible on all images.

    Reviewer #1 (Significance (Required)):

    *In the literature, there is a rich understanding of the molecular mechanisms of the cross-talk between cell-cell junctions, cell polarity complexes, and the organisation of the cytoskeleton. The authors are applauded for efforts to investigate whether there are common transcriptional downregulations of microtubule-related proteins that could potentially be key regulators of cellular polarisation. It is unfortunate that the work, as presented, is a series of modest observations, often the insight of which is overstated. Despite the analysis of plakins as a potential regulators of centrosome positioning between cell doublets, there is no mechanistic insight into a) how these plakins contribute to centrosome alignment asymmetry, or b) whether this is any way has an effect on true epithelial polarisation (beyond potential doublets on a micropattern). If significant development of mechanistic insight was added (requiring extensive additional experimentation, expected: 1-2 years of work), then the manuscript might be of interest to the cell biology community. *

    Authors: Our observations can be considered as modest but they are solid and novel and, to our point of view, significant. We acknowledge that the use of the term “epithelial polarity” instead of “asymmetric centrosome positioning” is an overstatement the impact of our observations and corrected it (including in the title). However, we think, based on previous works that are now better described in the revised version of our introduction, that the position of the centrosome in micropatterned cell doublets is a meaningful readout of the polarization of the organization of epithelial cells.

    It is true that we don’t provide the molecular and physical mechanism by which plakins affect centrosome position. And this would indeed deserve another complete study. However, since no previous work reported that plakins were involved in centrosome position, we think our work will be a valuable contribution to the field of cell polarity and to the recently rapidly growing field of plakins.

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

    Summary The new paper by Geay and colleagues studies epithelial cell polarity of triple negative breast cancer (TNBC) cell lines using a special H-shaped microculture device and confocal microscopy of centrosomes. Using a quantification method that calculates a polarity score, the polarity phenotype of each breast cancer cell line is associated to the corresponding transcriptome analyzed using an Affymetrix microarray platform. In this manner, expression of specific genes is correlated to the polarity score.

    In its second part, the study shifts its interest to the transdifferentiation process of EMT (epithelial-mesenchymal transition) and uses a published transcriptomic dataset based on human mammary epithelial cells that overexpress a series of oncogenic (Ras, TGF-beta) and EMT (ZEB1, ZEB2, TWIST1, E12) factors, and calculates an EMT score that is correlated to the expression of different genes identified in the published dataset. The interest in EMT is logical as EMT often correlates with the loss of epithelial cell polarity.

    Based on the two gene lists and their respective phenotypic correlation, known regulatory components of microtubule dynamics that can potentially regulate centrosomal position and thus epithelial cell polarity, are selected. Among these are genes encoding for components of desmosomes, the plakin family, that link membrane-based intercellular adhesions intracellularly to intermediate filaments, mainly cytokeratins, and indirectly with microtubules. Using traditional siRNA-based technology and an immortalized, non-malignant breast epithelial MCF10A cell line, silencing of specific plakin family mRNAs is shown to lead to polarity defects that correlate with concomitant high expression of the intermediate filament vimentin, the latter often used as a molecular marker of the EMT process. In other words, silencing of plakins leads to loss of breast epithelial cell polarity and gain of vimentin, a sign of enhanced EMT. This is the central observation of this study that is also captured in the title and it is not carried any further towards a mechanistic or deeper analysis. *

    Authors: We thank the reviewer for this fair and accurate description of our work

    *Major comments: I have two general or conceptual comments and one major technical comment:

    1. Unfortunately, the study does not provide an advance in terms of understanding the action of plakins as regulators of cell polarity or EMT. Both cellular processes are well characterized, and in the case of EMT, specific guidelines have been published that dictate the large number of complementary assays required for a proper assessment of EMT (see Yang, J., et al. Nat Rev Mol Cell Biol. 2020 Jun;21(6):341-352). *

    Authors: This is correct. We did not elucidate the underlying mechanism but we identified the involvement of plakins in the regulation of centrosome position in polarized epithelial cells. Note that our aim was not to reveal new EMT regulator, but to reveal new regulator of centrosome positioning. We only took advantage of EMT as a natural mechanism that involves the destabilisation of epithelial polarity and the reversal of centrosome position. The focus and the experimental strategy of our study are now better explained in the revised version of our introduction.

    *2) The plakins studied make the intracellular adaptor interface that links desmosomes to intermediate filaments, primarily cytokeratins. The paper does not even mention at all these two important epithelial protein networks, which I believe should have been studied in both TNBC and HME-EMT cell models. Furthermore, the paper tries to emphasize the regulation of microtubular networks because of their established importance in organizing proper centrosomal positioning. Yet, the presentation of results and the discussion appears rather confusing and unclear as to whether the data present any real effects of plakin expression manipulation on microtubules. It appears that such effects were not scored, which leaves the central aim of the project incomplete and raises issues that demand further and deeper analysis of the regulation of centrosome positioning by plakins. Can the centrosomal effects be completely indirect or bypasser effects due to the overall architectural change that epithelial cells undergo when their desmosomes lose their rigid coupling to cytokeratins? *

    Authors: It is true that we did not study at all the mechanism by which plakins affect centrosome position. And cytokeratins would definitely by on the top list for such a study. We we focused our transcriptomic analysis to microtubule-binding protein, since they are likely involved in the regulation of centrosome positioning. But we did not investigate in details the role of microtubules in the mispositioning of centrosome we found in plakin mutants. As stated by the reviewer, centrosome mispositioning might not result from a direct role of plakins on microtubules. The mechanism could definitely involve desmosomes, cytokeratins or many other cytoskeleton components. The exploration of all these possibilities should be the focus of future studies. Our work simply provides multiple and solid evidences for the implication of plakins in the regulation of centrosome positioning and open the way for interesting follow-up studies.

    *3) The classic siRNA-based method is used to silence plakin family mRNAs. This well-established technology today demands the use of multiple independent siRNAs per mRNA and also rescue experiments in order to confirm the absence of so-called off-target effects. *

    Authors: This is correct. We used two distinct siRNAs per targeted proteins. The effect on centrosome mispositioning were quite similar with both sequences (see Figure S2). We did not have time to confirm the absence of off-target effects by rescue experiment. This is missing indeed and unfortunately, we don’t have the human resources to perform those experiments. But we would like to stress out that a direct correlation between the level of expression of the tree plakins we tested and centrosome mispositioning was established in the first part of the study that was based on the natural variation of their expressions in 12 cells lines.

    Specific comments: I also enlist here some specific comments in the order of the figure presentation:

    *3) Fig. 1A lacks the images of Hs-578T and MCF10A cells. *

    Authors: This is correct. But MCF10A were already shown in our previous publication (Burute et al., Dev Cell, 2017). We did not want to insist on something already shown. Then we decided to show only 10 images as it would be odd to organise a panel with 11 images. Individual images are not so informative in this case, they are more illustrative and they are not so different from each other.

    *4) The data of Fig. 1C demonstrate score of 15-30% for the TNBC and "normal" epithelial cells. These data must be discussed in the context of the established literature on cell polarity. Is a 30% score anticipated for a polarized cell type? Is the difference between 30 and 20% significant in terms of the polarity of cells within a tissue? What would such scores be if one studied highly polarized cell monolayers on transwell filters? Is the H-shaped microsystem reliable? *

    Authors: This is a good remark and a fair concern. We can’t compare directly this “polarity score”, which is a metric about the position of centrosome, to the complete polarization of structures and signalling pathways in actual epithelial tissues in vivo. But we already studied the polarity of MDCK and MCF10A doublets (Burute et al, Dev Cell, 2017), which showed similar level of asymmetry in their centrosome position.

    It is also fair to doubt of the reliability of H-shaped micropatterned. In our revised introduction (see last paragraph), we have now listed all the features that made us believe that the polarized organisation of intercellular junctions and associated components in micropatterned cell doublets is relevant to the establishment of polarity in polarized epithelial tissues in vivo. The list of polarized components was based on two independent works (Rodriguez-Fraticeeli et al., J Cell Biol, 2012) (Burute et al., Dev Cell, 2017). In addition, it should be noted that we also previously reported the inversion of centrosome position in epithelial cell doublets during TGF-beta-induced EMT in MCF10A, and that we also observed this repositionging in vitro in 3D mammary gland cultured cells and in vivo in vivo, in mouse mammary gland epithelia and in developing mouse embryo at gastrulation (Burute et al., Dev Cell, 2017).

    *5) The gene expression data of the TNBCs or publicly available data for the same cell lines from TCGA should be used to generate a heat map that illustrates the positioning of the examined cells in the spectrum of luminal epithelial to claudin-low, mesenchymal breast cancer cells. *

    Authors: Such an analysis would be interesting indeed. Actually, a lot of information about the role of plakin in the maintenance of epithelial polarity could be extracted from the comparison of transcriptomic profiles of these various stages of EMT. But this is a bit beyond the scope of our study which was more focused on the consequences of these changes on centrosome position.

    *6) The 13 HME cell models used in Fig. 2A should be described in detail despite their earlier publication 11 years ago. This is important because the derived EMT scores are slightly counterintuitive: the parental HME cells are plotted as having a higher EMT score than the transformed HMEs expressing Ras or Twist1. How can this be explained? P53 is well established as an epithelial differentiation factor that counteracts EMT. Why does shp53 and especially combined with Ras overexpression not lead to EMT? I note that this cell model is listed as having epi and mes varieties. What are these and why are these important phenotypes not presented in the results? TGF-beta is presented in the results as a transcription factor, yet it is a secreted growth factor. What does TGF-beta mean? HME cells overexpress the cDNA for TGF-beta (which one? There are 3 TGF-beta genes)or were the cell cultured in the presence of this cytokine? *

    Authors: These are interesting comments. Actually, one of the important observations of this earlier study was that mice over-expressing Ras alone or Twist alone in mammary tissues, either during embryonic development or later during mammary development induced by lactation, did not form invasive tumours. The expression of Ras induced low grade splenic lymphomas as well as anal and oral papillomas but they never progressed to the malignant stage. However, the combination of Ras and Twist expression had dramatic effects on the reduction of mice survival due to the formation of multifocal breast carcinomas with metaplastic features. So the absence of increase of the EMT score upon the overexpression of Ras or Twist alone is not so counterintuitive. But we can’t really explain how cells became “more epithelial” though. We think that it would be long and not so conclusive to enter into those details in the main text.

            Cells silenced for p53, to resist from oncogene-induced senescence and apoptosis, and over-expressing Ras could express or not EpCAM and thus were sorted in EpCAM positive (epi) or EpCAM negative (mes). In some conditions, TGF-beta was added to cells to induce EMT. The combinations of these various treatments induced more or less aggressive transformations that are described in this earlier study but we think it would take too long to describe them here.

    In the end, what mattered for our study, was that this set of cell lines allowed us to explore a broad range of EMT scores, which we could correlate to variations of transcriptomic profiles.

    *7) Minor semantic comment: does Fig.2B show collagen V or collagen XV? Related to this, the article has abundant typographical errors. *

    Authors: It was collagen V. We checked for other typos and hope to have corrected them.

    *8) Fig. 4: based on the major comment, this experiment requires analysis of rescue clones. *

    Authors: We fully agree, these experiments are missing indeed. Unfortunately, we don’t have the human resources to perform those experiments. However, it should be noted that the specificity of the target is somehow supported by the observation of the exact same phenotypes upon the use of another siRNA sequence for each plakins (Figure S2). In addition, we would like to stress out that a direct correlation between the level of expression of the tree plakins we tested and centrosome mispositioning was established in the first part of the study that was based on the natural variation of their expressions in 12 cells lines (Figure 1).

    *9) Fig. 5C: the vimentin microscopy needs to be complemented with full EMT analysis using both microscopic and protein expression assays (see major comment). More importantly, desmosomal and cytokeratin organization analysis is missing. *

    Authors: We agree that an immunostaining of vimentin is way too preliminary to conclude about an actual induction of EMT. Hence our tempered conclusion about the “suggestion that cells might be engaged in a form of EMT”. As also mentionned by reviewer #1 a full characgterization of the EMT state of these cells would require a long list of measurements, including the quantification of EMT-related transcripts and other structural analysis like desmosome and cytokeratins. But we don’t have the manpower to perform these experiments. Considering the broad interest of our community for the induction of EMT we thought that these observations were sufficiently interesting to be reported although they were somehow distant from the focus of our study on centrosome positioning.

    *10) Fig. 5C: In the desmoplakin and periplakin knock-down experiments the cells stained for vimentin appear to have their vimentin "baskets" rather well polarized. Is this true or my impression based on the few cells illustrated in the images? If the cytoskeleton is polarized, what does one mean with loss of cell polarity? Is centrosomal polarity change associated with mesenchymal (back to front) polarity gain? If this is true can polarity be established by studying only 2 cells in the H-shaped microcultures? Is it not more relevant to allow cells build a cohort of inter-adherent cells? *

    Authors: This is an interesting observation and a thoughtful analysis. And indeed, it can be seen in the quantification of polarity indices of periplakin knocked-down cells (see Figure 4E and figure S2) that the distribution seems to contain two distributions, one of epithelial-like orientation (values closed to +1) and one of reversed orientation (values closed to -1), toward the ECM, similar to our previous description of polarity reversal during EMT (Burute et al, Dev Cell, 2017). This suggest that indeed the knockdown of periplakin might not only impair the epithelial apico-basal polarity but also promote mesenchymal front-back polarity. Although interesting, we found it a bit too speculative to be stated in our revised version.

    *Reviewer #2 (Significance (Required)): *

    Significance: The paper provides a quantitative analysis of single cell polarity and gene expression-based EMT and identifies plakin gene family members as potential regulators of cell polarity. If this finding can be substantiated via mechanistic work it will make an important contribution to epithelial cell biology. *

    General assessment: As explained above, the paper is in a preliminary state, as it describes an observation that demands further analysis. Key cell biological constituents (desmosomes, cytokeratins) have not been included in the analysis. Specific key figure data are presented without explanation for the non-specialist, especially those data that have been generated based on older publications by some of the authors. *

    Advance: At the present state, the paper does not make any advance but describes potentially interesting observations. *

    Audience: This paper can stimulate interest in the broader field of cell biology and definitely more to the cell polarity and EMT sub-fields. *

    Authors: It is true that we don’t provide the molecular and physical mechanism by which plakins affect centrosome position. And this deserves indeed further characterisation. However, our work provides multiple and solid evidences for the implication of plakins in the regulation of centrosome positioning in epithelial cells and thus opens the way for interesting follow-up studies. Since no previous work reported this role of plakins, we think our work will be a valuable contribution to the field of cell polarity and to the recently rapidly growing field of plakins.

    *My field of expertise: I study signal transduction and transcriptional mechanisms that regulate the EMT and its association with cell proliferation in cancer cells. I have also specialized on studying dynamics of cytoskeletal assembly and architectural cell organization.

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

    In this article the authors have analyzed the genes related to epithelial cell polarity and report the relevance of the desmosomal proteins epiplakin, desmoplakin and periplakin in this process. These genes are downregulated in cells that have lost cell polarity and their lack of expression correlates with the emergence of an EMT program. Moreover, interference in the expression of these proteins increase vimentin and likely other mesenchymal markers. The experiments are very neatly done, with all the appropriate controls; the methodology is adequate, the figures are well designed and are reader-friendly and the results have some interest. Therefore, I do not have objections related to these issues, other than two minor question indicated below. *

    Authors: We thank the reviewer for this positive assessment of our work

    *- Other EMT markers should be easily assessed in the cells transfected with the plakins shRNAs to analyze the extent of EMT in these cells. *

    Authors: We agree that an immunostaining of vimentin is way too preliminary to conclude about an actual induction of EMT. Hence our tempered conclusion about the “suggestion that cells might be engaged in a form of EMT”. As also mentionned by reviewer #1 a full characterization of the EMT state of these cells would require a long list of measurements, including the quantification of EMT-related transcripts and other structural analysis like desmosome and cytokeratins. But we don’t have the manpower to perform these experiments. Considering the broad interest of our community for the induction of EMT we thought that these observations were sufficiently interesting to be reported although they were somehow distant from the focus of our study on centrosome positioning.

    *- It would be interesting if the article is reviewed by a scientist with a deeper knowledge in EMT because the text contains some inaccuracies related to this process and the main references are outdated. *

    Authors: It is unfortunate that the reviewer was not more specific in his/her assessment. There are lots of references in the field of EMT. Not so many are related to the polarized organization of cells and we tried to cited those we found significant. We have added more recent references in the revised version of our introduction. We hope this will be satisfactory but we would be happy to complement this list.

    *Reviewer #3 (Significance (Required)):

    However, the significance of the conclusions is very limited. The relevance of desmosomes in cell polarity was described time ago by the Fuchs' group (see Lechler T, Fuchs E, J Cell Biol 2007, 176, 147-154); since then, this topic has been investigated by many other labs. For a more recent work see "Desmosomes polarize and integrate chemical and mechanical signaling to govern epidermal tissue form and function" Broussard et al, Curr Biol 2021, 31, 3275-3291. *

    Authors: This is correct. The role of desmosome in the establishment and maintenance of the apical pole of epithelial has been well established. However, their role in the positioning of centrosome is much less clear.

    Please note that the paper by Lechler and Fuchs is not about epithelial polarity. It describes the loss of astral organisation of microtubules in differentiating epidermal cells forming desmosomes thanks to the recruitment of ninein to desmosoems by desmoplakin.

    Please also note that the other study by the group of Kathleen Green is about the role of desmoplakin in ensuring distinct mechanical states in the apical and basal pole of epidermal cells. It is not related to the organisation of the microtubules, nor is it related to the position of the centrosome. So it is unclear to us how these works limit the significance of our findings about the role of plakins in the control of centrosome position and the establishment of apico-basal polarity. We were happy to include them in the revised version of our discussion anyway.

    Furthermore, our work is about three distinct plakins: periplakin, epiplakin and desmoplakin. Although they all localise to desmosomes their specific roles in the establishment and maintenance of epithelial polarity has not yet been established (as detailed in the general comments we wrote in the opening of this letter and in the revised version of our discussion). In addition, their specific roles should be distinguished from the multiple roles of desmosome in cell polarity, which involve inter-cellular junctions and connections to various inner cytoskeleton networks.

    So, although we acknowledge that a mechanistic understanding would significantly increase the strength of our study, we still believe that the demonstration of the involvement of these three plakins in the regulation of centrosome position in polarized epithelial cells is novel and significant.

    *Therefore, the authors need to analyze the mechanism with a greater detail if they want to contribute to the advance of this field. As a possible suggestion, they might use their plakin shRNA-transfected cells to investigate the signaling pathways that are altered, to transfect different desmoplakin mutants and describe their effects. *

    Related to desmosome alterations and EMT, this has also been indirectly concluded in an article quoted by the authors (Chun and Hanahan). This might be also studied by the authors assessing if the main transcriptional factors related to EMT are altered in these cells. *

    Authors: We thank the reviewer for these constructive suggestions to deepen our investigation. The identification of these pathways could definitely highlight the mechanisms involved in the regulation of centrosome positioning. However, this is somehow beyond the scope of this study which was focused on the identification of regulators of centrosome asymmetric positioning in polarized epithelial cells. Counterintuitively, molecular motors did not seem to be involved. But several plakins were revealed. Further studies are now required to understand how they impact centrosome position.

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    Referee #3

    Evidence, reproducibility and clarity

    In this article the authors have analyzed the genes related to epithelial cell polarity and report the relevance of the desmosomal proteins epiplakin, desmoplakin and periplakin in this process. These genes are downregulated in cells that have lost cell polarity and their lack of expression correlates with the emergence of a n EMT program. Moreover, interference in the expression of these proteins increase vimentin and likely other mesenchymal markers. The experiments are very neatly done, with all the appropriate controls; the methodology is adequate, the figures are well designed and are reader-friendly and the results have some interest. Therefore, I do not have objections related to these issues, other than two minor question indicated below.

    • Other EMT markers should be easily assessed in the cells transfected with the plakins shRNAs to analyze the extent of EMT in these cells.

    • It would be interesting if the article is reviewed by a scientist with a deeper knowledge in EMT because the text contains some inaccuracies related to this process and the main references are outdated.

    Significance

    However, the significance of the conclusions is very limited. The relevance of desmosomes in cell polarity was described time ago by the Fuchs' group (see Lechler T, Fuchs E, J Cell Biol 2007, 176, 147-154); since then, this topic has been investigated by many other labs. For a more recent work see "Desmosomes polarize and integrate chemical and mechanical signaling to govern epidermal tissue form and function" Broussard et al, Curr Biol 2021, 31, 3275-3291.

    • Therefore, the authors need to analyze the mechanism with a greater detail if they want to contribute to the advance of this field. As a possible suggestion, they might use their plakin shRNA-transfected cells to investigate the signaling pathways that are altered, to transfect different desmoplakin mutants and describe their effects.

    • Related to desmosome alterations and EMT, this has also been indirectly concluded in an article quoted by the authors (Chun and Hanahan). This might be also studied by the authors assessing if the main transcriptional factors related to EMT are altered in these cells.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The new paper by Geay and colleagues studies epithelial cell polarity of triple negative breast cancer (TNBC) cell lines using a special H-shaped microculture device and confocal microscopy of centrosomes. Using a quantification method that calculates a polarity score, the polarity phenotype of each breast cancer cell line is associated to the corresponding transcriptome analyzed using an Affymetrix microarray platform. In this manner, expression of specific genes is correlated to the polarity score.

    In its second part, the study shifts its interest to the transdifferentiation process of EMT (epithelial-mesenchymal transition) and uses a published transcriptomic dataset based on human mammary epithelial cells that overexpress a series of oncogenic (Ras, TGF-beta) and EMT (ZEB1, ZEB2, TWIST1, E12) factors, and calculates an EMT score that is correlated to the expression of different genes identified in the published dataset. The interest in EMT is logical as EMT often correlates with the loss of epithelial cell polarity.

    Based on the two gene lists and their respective phenotypic correlation, known regulatory components of microtubule dynamics that can potentially regulate centrosomal position and thus epithelial cell polarity, are selected. Among these are genes encoding for components of desmosomes, the plakin family, that link membrane-based intercellular adhesions intracellularly to intermediate filaments, mainly cytokeratins, and indirectly with microtubules. Using traditional siRNA-based technology and an immortalized, non-malignant breast epithelial MCF10A cell line, silencing of specific plakin family mRNAs is shown to lead to polarity defects that correlate with concomitant high expression of the intermediate filament vimentin, the latter often used as a molecular marker of the EMT process. In other words, silencing of plakins leads to loss of breast epithelial cell polarity and gain of vimentin, a sign of enhanced EMT. This is the central observation of this study that is also captured in the title and it is not carried any further towards a mechanistic or deeper analysis.

    Major comments:

    I have two general or conceptual comments and one major technical comment:

    1. Unfortunately, the study does not provide an advance in terms of understanding the action of plakins as regulators of cell polarity or EMT. Both cellular processes are well characterized, and in the case of EMT, specific guidelines have been published that dictate the large number of complementary assays required for a proper assessment of EMT (see Yang, J., et al. Nat Rev Mol Cell Biol. 2020 Jun;21(6):341-352).

    2. The plakins studied make the intracellular adaptor interface that links desmosomes to intermediate filaments, primarily cytokeratins. The paper does not even mention at all these two important epithelial protein networks, which I believe should have been studied in both TNBC and HME-EMT cell models. Furthermore, the paper tries to emphasize the regulation of microtubular networks because of their established importance in organizing proper centrosomal positioning. Yet, the presentation of results and the discussion appears rather confusing and unclear as to whether the data present any real effects of plakin expression manipulation on microtubules. It appears that such effects were not scored, which leaves the central aim of the project incomplete and raises issues that demand further and deeper analysis of the regulation of centrosome positioning by plakins. Can the centrosomal effects be completely indirect or bypasser effects due to the overall architectural change that epithelial cells undergo when their desmosomes lose their rigid coupling to cytokeratins?

    3. The classic siRNA-based method is used to silence plakin family mRNAs. This well-established technology today demands the use of multiple independent siRNAs per mRNA and also rescue experiments in order to confirm the absence of so-called off-target effects.

    Specific comments:

    I also enlist here some specific comments in the order of the figure presentation:

    1. Fig. 1A lacks the images of Hs-578T and MCF10A cells.

    2. The data of Fig. 1C demonstrate score of 15-30% for the TNBC and "normal" epithelial cells. These data must be discussed in the context of the established literature on cell polarity. Is a 30% score anticipated for a polarized cell type? Is the difference between 30 and 20% significant in terms of the polarity of cells within a tissue? What would such scores be if one studied highly polarized cell monolayers on transwell filters? Is the H-shaped microsystem reliable?

    3. The gene expression data of the TNBCs or publicly available data for the same cell lines from TCGA should be used to generate a heat map that illustrates the positioning of the examined cells in the spectrum of luminal epithelial to claudin-low, mesenchymal breast cancer cells.

    4. The 13 HME cell models used in Fig. 2A should be described in detail despite their earlier publication 11 years ago. This is important because the derived EMT scores are slightly counterintuitive: the parental HME cells are plotted as having a higher EMT score than the transformed HMEs expressing Ras or Twist1. How can this be explained? P53 is well established as an epithelial differentiation factor that counteracts EMT. Why does shp53 and especially combined with Ras overexpression not lead to EMT? I note that this cell model is listed as having epi and mes varieties. What are these and why are these important phenotypes not presented in the results? TGF-beta is presented in the results as a transcription factor, yet it is a secreted growth factor. What does TGF-beta mean? HME cells overexpress the cDNA for TGF-beta (which one? There are 3 TGF-beta genes)or were the cell cultured in the presence of this cytokine?

    5. Minor semantic comment: does Fig.2B show collagen V or collagen XV? Related to this, the article has abundant typographical errors.

    6. Fig. 4: based on the major comment, this experiment requires analysis of rescue clones.

    7. Fig. 5C: the vimentin microscopy needs to be complemented with full EMT analysis using both microscopic and protein expression assays (see major comment). More importantly, desmosomal and cytokeratin organization analysis is missing.

    8. Fig. 5C: In the desmoplakin and periplakin knock-down experiments the cells stained for vimentin appear to have their vimentin "baskets" rather well polarized. Is this true or my impression based on the few cells illustrated in the images? If the cytoskeleton is polarized, what does one mean with loss of cell polarity? Is centrosomal polarity change associated with mesenchymal (back to front) polarity gain? If this is true can polarity be established by studying only 2 cells in the H-shaped microcultures? Is it not more relevant to allow cells build a cohort of inter-adherent cells?

    Significance

    Significance: The paper provides a quantitative analysis of single cell polarity and gene expression-based EMT and identifies plakin gene family members as potential regulators of cell polarity. If this finding can be substantiated via mechanistic work it will make an important contribution to epithelial cell biology.

    General assessment: As explained above, the paper is in a preliminary state, as it describes an observation that demands further analysis. Key cell biological constituents (desmosomes, cytokeratins) have not been included in the analysis. Specific key figure data are presented without explanation for the non-specialist, especially those data that have been generated based on older publications by some of the authors.

    Advance: At the present state, the paper does not make any advance but describes potentially interesting observations.

    Audience: This paper can stimulate interest in the broader field of cell biology and definitely more to the cell polarity and EMT sub-fields.

    My field of expertise: I study signal transduction and transcriptional mechanisms that regulate the EMT and its association with cell proliferation in cancer cells. I have also specialized on studying dynamics of cytoskeletal assembly and architectural cell organization.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Geay and colleagues examine potential regulators of centrosome positioning in an immortalised breast cell line in vitro on micropatterns that promote cell doublet formation. The authors mine expression data from breast cancer cell lines in vitro to identify microtubule-related transcripts that are potentially downregulated in cells with a mesenchymal phenotype. The authors identify some Plakin proteins, which upon depletion, are reported to change centrosome positioning relative to junctions. The authors propose that plakins are involved in the maintenance of epithelial polarity.

    Major comments:

    I applaud the authors for attempting to identify novel regulation of epithelial polarity. However, I am sorry to say that this manuscript is overtly preliminary. It is a collection of observations without any mechanistic insight (described below). Despite what I write below, I apologise in that these shortcomings as so extensive that I cannot recommend experiments that would 'fix holes', without essentially writing an entirely new project. Even after addressing the points below, I think it unlikely that the observations would make a coherent, mechanistic contribution to the field of epithelial polarity. I do not like to give reviews like this, but unfortunately, the submission of such preliminary works puts us in this position.

    1. 'Epithelial polarity' Throughout manuscript the authors refer to a 'polarity score' and the term 'epithelial polarity' when what they have actually measured is a specific angle of orientation of centrosomes in cell doublets in vitro. This is an overstatement and adds confusion. The term 'epithelial polarity' has overtones of a polarised epithelium, which such doublets do not model. There is no mechanistic investigation into how this polarity score relates to the ability to form a polarised epithelial monolayer, with apical-basal polarity orientations, either a monolayer on a substrate or a monolayer surrounding a single central lumen, such as these MCF10A cells are often used for in 3-dimensional culture. I suggest that the authors simply mention what they actually measure (and in their own words): "coordination of the centrosome along the nucleus-junction axis."

    2. In Figure 1A-C, cell doublets are reported and apparently quantified to measure a 'polarity score', which is the angle of orientation of centrosomes in cell doublets. Yet, there is no clear information that explains how the cutoff for what defines this polarity score is generated (e.g. why is the cutoff point chosen to be where it is?), or what it means for epithelial polarity (e.g. why is this cutoff point important to be at that site?). Moreover, there is no indication that these cells actually form connected doublets. Labelling and quantitation of potentially connected cells is absent. Do these actually form junctions to the same extent, such that any differences have been exhaustively excluded to be only from the centrosome orientation, rather than cell spreading and cell-cell contact differences (that would alter geometry)? In addition, statistical analysis for part C is missing.

    3. Fig 1D, 2A,B present select example genes correlated with either polarity score or EMT score (Fig 1D, 2B). It is unclear what insight providing select genes from many that are changed provides. In Fig 2A, an apparent EMT score (seemingly derived from mining of existing expression data not from this laboratory) is provided, ranked by an EMT. No description is provided for what these alterations are (e.g. what is a 'HME_Ras_Twist1E12_TGFb' sample?). Further, what this is supposed to indicate as a mechanistic insight is unclear.

    4. Figure 3 is highly preliminary. The entirety of Figure 3 is a correlation plot between EMT score and polarity score for microtubule-related transcripts. The authors state:

    "The graph showed an overall negative trend, which means that many genes were positively correlated with an EMT in HME were instead negatively correlated with epithelial polarity of TNBCs (Figure 3). This was expected and confirmed that the progression along EMT is associated with a loss of epithelial polarity."

    No statistical analysis is presented, no correlation scores and indication of robustness is provided. It is unclear how this provides any mechanistic insight. The authors themselves state that this association is expected.

    Moreover, the authors state "Interestingly, three plakins, namely epiplakin (EPPK1), desmoplakin (DSP) and periplakin (PPL) all appeared as clear outliers (Figure 3)."

    How is an outlier defined & why is this clear? Is the association of these key cell-adhesion molecules with an epithelial cell state novel or known?

    1. Figure 4. The authors perform siRNA-mediated depletion of Desmoplakin, Epiplakin and Periplakin in MCF10A cells. The authors report, "Interestingly, knocked-down cells in culture displayed abnormal shapes, being more elongated and less cohesive (Figure 4C)."

    No quantitation of such changes are provided. Moreover, cells with KD appeared to be at lower density. Can the authors exclude that these are not merely density-dependent effects.

    1. Throughout the work, the polarity index is reported from plakin depletion conditions with data from a reported 3 independent experiments seemingly pooled (no indication of graph of which independent experiment each data point comes from). Is the statistical analysis performed (missing in Fig 4E, present in Fig 5A-C, S2, S3) from pooled data? If so, this is in appropriate and should be from the averages of independent experiments, to understand batch effects. If not from pooled data, please alter graphs to display this appropriately.

    2. Figure 5A. It is unclear how F-actin is measure in the images. Is F-actin labelling a truly representative proxy for junction length?

    3. Fig 5C. Why are images of vimentin now provided not on micropatterns? The labelling of vimentin in siPeriplakin cells does not look appropriately controlled for by the other cell conditions. siPeriplakin is clearly at the edge of a colon, whereas this is not clear whether an appropriate region is labelled in the other conditions.

    Significance

    In the literature, there is a rich understanding of the molecular mechanisms of the cross-talk between cell-cell junctions, cell polarity complexes, and the organisation of the cytoskeleton. The authors are applauded for efforts to investigate whether there are common transcriptional downregulations of microtubule-related proteins that could potentially be key regulators of cellular polarisation. It is unfortunate that the work, as presented, is a series of modest observations, often the insight of which is overstated. Despite the analysis of plakins as a potential regulators of centrosome positioning between cell doublets, there is no mechanistic insight into a) how these plakins contribute to centrosome alignment asymmetry, or b) whether this is any way has an effect on true epithelial polarisation (beyond potential doublets on a micropattern). If significant development of mechanistic insight was added (requiring extensive additional experimentation, expected: 1-2 years of work), then the manuscript might be of interest to the cell biology community.

  5. Version published to 10.1101/2023.10.16.562500v2 on bioRxiv
  6. Version published to 10.1101/2023.10.16.562500v1 on bioRxiv