Condensation tendency and planar isotropic actin gradient induce radial alignment in confined monolayers

  1. Tianfa Xie
  2. Sarah R St Pierre
  3. Nonthakorn Olaranont
  4. Lauren E Brown
  5. Min Wu
  6. Yubing Sun

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

A monolayer of highly motile cells can establish long-range orientational order, which can be explained by hydrodynamic theory of active gels and fluids. However, it is less clear how cell shape changes and rearrangement are governed when the monolayer is in mechanical equilibrium states when cell motility diminishes. In this work, we report that rat embryonic fibroblasts (REF), when confined in circular mesoscale patterns on rigid substrates, can transition from the spindle shapes to more compact morphologies. Cells align radially only at the pattern boundary when they are in the mechanical equilibrium. This radial alignment disappears when cell contractility or cell-cell adhesion is reduced. Unlike monolayers of spindle-like cells such as NIH-3T3 fibroblasts with minimal intercellular interactions or epithelial cells like Madin-Darby canine kidney (MDCK) with strong cortical actin network, confined REF monolayers present an actin gradient with isotropic meshwork, suggesting the existence of a stiffness gradient. In addition, the REF cells tend to condense on soft substrates, a collective cell behavior we refer to as the ‘condensation tendency’. This condensation tendency, together with geometrical confinement, induces tensile prestretch (i.e. an isotropic stretch that causes tissue to contract when released) to the confined monolayer. By developing a Voronoi-cell model, we demonstrate that the combined global tissue prestretch and cell stiffness differential between the inner and boundary cells can sufficiently define the cell radial alignment at the pattern boundary.

Article activity feed

  1. Version published to 10.7554/elife.60381 on eLife
  2. eLife

    ###Reviewer #3:

    The authors study the effect of confinement on the alignment of REF cells confined within circular micropatterned islands. They observed that the cells are aligned perpendicularly to the boundary after 48h, contrary to other elongated cells such as NIH-3T3. After testing several subclones of that cell line, they identified cell contractility and cell-cell adhesion that affect the organization of the cells in the circular patterns. They confirmed this using drugs that affect contractility and disrupt cell adhesion. Then they compared their results to a continuum model and to a voronoi model.

    The science is interesting. Many cell types are elongated and do align with their neighbors. The fact that these cells align perpendicularly to a boundary is curious, and deserved to be studied in depth. 3 similar papers came out on arxiv from the Roux group. They should be discussed in the manuscript and cited.

    It is not clear what is the "condensation" process the authors are referring to and how this is related to the boundary alignment of the REF cells. Please, read the work of trepat et al on active dewetting published in 2018. I do not know what the author means by tendency. Some it condensed, sometimes It does not? IT is not a scientific term. I would advise choosing different wording to explain their results. Condensation is the first word in the title of the manuscript, still it appears for the first time in the text on page 18, and is poorly defined. It is never well explained and the 2 terms always come up together, condensation and tendency, like if the author does not know themselves what to call what they are observing.

    There is a lot of data, analysis and model, but it is very confused, not well organized and poorly presented, which prevents me from judging the quality of the interpretations. The authors chose to show all the analysis they could do in the figures, and therefore there is no clear take-home message. Are all those plots necessary?

    It was a very difficult paper to read. Often, terms like nematic, or symmetry are misused. Such words have a very specific meaning, in particular for liquid crystal physisicst, which are one of the targeted audience for this paper. The figures are not clear. They at the same time put too much information and not enough. There are too many graphs, I don't know which one is important. Please, plot the 2 cells types in the same graph instead of showing one graph/cell type. At the same time, there is often not enough information to understand what the authors are plotting, and what is the take-away message.

    Below, I have specific comments about the text, not so much about the science. Again, I found it very hard to read and understand, hence I am not able to judge the quality of the research at that point.

    Specific comments about the figures: Fig 3: What is the unit of the heat maps? Please add fluorescent image, and average for the second row, and for the plot, please, add a label "normalized mean intensity" of what?

    I do not understand Fig 4. The captions just reads the labels of the plots, it does not tell me the results, nor the relevance.the is no information in the caption, please revise.

    What is the main result of Fig5? The title could not be more vague: "Voronoi cell modeling predicts REF 2c cell behaviors in circular pattern.". Please give specific titles to your figures that help the reader understand the take-away message. Please change the contrast of Fig. 5A. All the disks look black to me. I have difficulties trusting statistical analysis. The top right plot of fig 5C looks totally flat to me. Why is there sometimesa statistical analysis and sometimes not? ( %B 1,2,4 and %C1 have no statistical analysis).

    Same critics for Figure 6.

    About the abstract: The terminology is vague and confusing, which I think that the authors have not fully characterized the connection between their experiments and the physics of liquid crystals. examples: "to form nematic symmetry" "to form a new type of symmetry" "new symmetry?" changing boundary condition does not mean you are changing the symmetry of the liquid crystal...

    Strong adhesive interaction... MDCK also have strong adhesive interactions, therefore the comparison is not adequate, please revise.

    What does "condensation tendency" mean? What does "prestrech" in the last sentence of the abstract mean? Is the tissue under stretch? There is no reference to stretch in the abstract before that.

    Comments about the introduction: The introduction is scattered, very confusing as it mixes results from a broad range of model systems. For example in 4 successive sentences, we have: adipocytes, then fish then reconstituted asters, then back to muscle cells. This looks like a laundry list... Same thing in the next paragraph: neural crest cells, mesenchymal stem cells, chondrocytes, At this point, it is not clear what cells types the authors are studying and why it is relevant to all the others cited in the introduction.

    Cell condensation is not "unique" to their cell types. MDA-MB-231 also do that ( ref: TRepat et al, Active wetting of epithelial tissues, 2018).

    "to robustly self-organize in polarized organization", please rephrase

    "mechanical variable have been used to describe the mechanical behavior of a cell monolayer", please rephrahe, this is way too vague. What are you trying to say?

    Why epitheial-like? Why not just epithelial? Are these cells different?

    What does "presented cytoskeleton" mean?

    3T3 cells are not incompressible. No cell types are. They divide all the time.

    You can have radial alignment in a nematic liquid crystal, it is called homeotropic anchoring. It has nothing to do with the symmetry of the liquid crystaline units.

    Condensation driven by chemotaxis? I never heard that. See again TRepat et al 2018. The cells are confined in a similar circular island, there is no chemotaxis.

    References were not properly cited. As an example ref [11] does not talk about the effect of confinement at all.

    About the methods: Manual tracking is passe. There are robust methods to automatically track cells. You are already segmenting the tissue, why not tracking the cells automatically this way?

    "The average speed for each cell was calculated as the total migration length of each cell divided by the total time". So if I track the cells for long enough and they diffuse randomly, the average speed is 0? Does not really make sense. For how long were the cells tracked? Are all the trajectories the same length?

    About the model: What other types of stress were neglected in the model and why? Especially, if you are trying to model a nematic liquid crystal, why not take into account the nematic elastic stress?

    Why nematic-like? This is confusing as is much of the terminology used in this manuscript.

  3. eLife

    ###Reviewer #2:

    This article reports the radial alignment of rat embryonic fibroblasts at the periphery of circular confinement patterns. The authors experimentally isolate that contractility, adhesion and stiffness gradient are necessary to obtain this alignment. They further devise continuum and discrete models, with only two free parameters, to describe the mechanical origin of such cellular arrangements.

    The article is an interesting contribution to the field, with the discussion and conclusion well supported by the experimental data. It is further well written, with a good logic.

    1. The authors should explain (e.g., in an appendix) how they solve Eqs.(7-9) and how they run their Voronoi simulations (or indicate which solver/package they use if those already exist).

    2. A movie showing the formation of the radially aligned cell pattern would be a good addition, even if the dynamics are not discussed in the article. The x,y,t axes should be labelled (with units) in Fig.1-Supp.1.

    3. p.17 l.3, "stiffnesses" instead of "substrates"?

    4. p.20 l.7, the authors should better explain how Fig.1-Supp.4 supports a homogeneous isotropic contractility.

    5. The authors should show some of the images used to extract actin fibers structure (or are these shown in Fig.3?). Is Fig.4-Supp.1 obtained for REF 2c?

    6. p.24 l3, the authors may comment on how stiffness anisotropy could be incorporated in their model to explain inner cells' circumferential alignment. The author should plot the structure parameter (k_h) vs radial distance instead of giving a table (Fig.4-Supp.1 and Fig.6-Supp.1); they should use the same origin (the center of the circle) for the radial distance in the ring experiments (x-axis in Fig.6B and Fig.6-Supp.1A vs x-axis in Fig.7 and Fig.7-Supp.1) to facilitate comparisons.

    7. The authors should clarify what they mean by "clear boundary junctions" (p.18 l.9) when describing Fig.2D, which is challenging to discern.

    8. In Fig.4, are the authors showing the strain or the stretch ratio? It would help to start the y-axis at 0 in Figs.4A-B. At which distance are the radial strain and stress evaluated in Figs.4C-D? Are the pre-stretch ratio and stiffness gradient challenging to evaluate from the experiments (p.20 l.4)? Can the authors comment on the values needed for these model parameters to see radial alignment in the simulations? Are they realistic when compared to the experimental data?

  4. eLife

    ###Reviewer #1:

    The manuscript by Xie et al combines an impressive array of experimental and modeling approaches to study cell morphological changes due to stiffness heterogeneities and contractility.

    1. The assumption of a purely elastic process needs substantiation. Fig. 1A shows a dramatic increase in the number of REF2c cells from 24 to 48 hours, suggesting that cells are proliferating. This, together with continuous remodeling of cell-cell contacts, would result in deformations that dissipate elastic energy. Neither modeling approach accounts for this. It would be important for authors to incorporate these behaviors, or to provide evidence that cell proliferation and remodeling are unimportant, and similar between the three cell populations being compared.

    2. The assumption that contractility is uniform needs to be substantiated. Work cited (Tambe et al) shows on the contrary that collective cell behaviors exhibit highly heterogeneous active stresses. Experimentally, there are a few potential ways at this. Authors could use the stiffer (1 MPa) micro post cultures, which recreate radial alignment seen on micropatterned PDMS islands, and compute force variations from post deflection. Alternatively, authors could perform short time lapse experiments to measure deformations following treatment with blebbistatin or Y27632. Yet another option would be to perform staining for contractile proteins such as phospho-myosin light chain, GTP-bound RhoA, or others, to confirm they are uniformly distributed despite the heterogeneity of F-actin (although this reviewer is skeptical that such experiments would reveal uniform contractility when F-actin is nonuniform). Finally, if no experimental support is possible, then authors could turn to model simulations to test whether spatial heterogeneities in contractility alter the overall behavior of the system (although, again, this reviewer is skeptical that such simulations would suggest the heterogeneity of contraction is unimportant). In addition to either modeling or experimental support for the assumption that contractility is uniform, authors should provide examples from the literature on related systems that support this assumption.

    3. The importance of a stiffness gradient in the cell population is one of the key aspects of this work. However, evidence for the existence of such a gradient is provided only by staining for F-actin, which is insufficient. While F-actin is indeed a key cytoskeletal component in defining the stiffness of cells, the link between intensity of staining and stiffness needs to be proven. Only a single reference is provided, which focused on one specific cancer cell line and the role of stress fibers - a specific configuration of F-actin together with myosin - in stiffening the cell. Moreover, given that F-actin interacts with nonmuscle myosin to form the key contractile machinery of most cell types, heterogeneity in F-actin likely implies heterogeneity in contractility as well. There are also concerns with the measurement of F-actin abundance, including need for statistics on the spatial distribution, and to normalize per cell to reflect variations in F-actin as opposed to simply variations in cell density, which are also present (Fig. 1A). Finally, the F-actin gradient is only shown and quantified when intensities are summed over many samples. It would be important to demonstrate a significant gradient within individual samples, and how it varies across samples.

    4. Greater integration between modeling and experiment would strengthen the manuscript. This is particularly true of the continuum model, where it is nontrivial to relate strain and stress to cell shape changes, given that cell shape is not simply an affine elastic deformation owing to stresses acting on it, but instead a response to stresses integrated with cell autonomous behaviors. There is a large body of literature on the alignment of cells relative to the direction of applied static or dynamic stretch. This mechano-responsivity that dictates cell shape is not considered in the present study. Even without considering these complicating cell behaviors, it is not clear how the magnitude of stress or strain relate to the change in cell shape. In addition, authors would ideally make use of the models to pinpoint what underlies the distinct polarization phenotypes between REF2c, REF11, and 3T3 cell types.

    5. The importance of cell-cell adhesion is another crux of the story, pointing to differences underlying the various polarization phenotypes. However, the only experimental support for this is via treatment with a calcium chelator, EGTA. Only one reference is provided for this method (#35, Chen et al), yet Chen et al appear not to have used EGTA at all, and instead disrupted E-Cadherin using neutralizing antibodies. This is a much more specific and direct approach that the authors of the present study should consider in place of EGTA. In the absence of this or similarly targeted approaches (RNAi, etc), the authors should include control experiments that demonstrate this rather broad perturbation does not alter contractility or cell-substrate interactions. This could be done at least in part, by using the traction force measurement system the authors have devised. It is particularly important to do so given the importance of calcium for cytoskeletal contraction via calmodulin. A second experiment authors could supplement this with is pharmacologic inhibition of calcium-depdendent contractility, with the hope/expectation that calmodulin-mediated contractility does not predominate this system. Even with these experiments, however, authors need to provide support from published work that this method of disrupting cell-cell adhesion is well established.

    6. The system is quite artificial with respect to in vivo conditions in most contexts. This on its own is not a limitation, as such approaches can still be used to reveal fundamental insights into the mechanisms of cell behaviors and interactions, employing approaches that are not feasible in vivo. However, it is important to tie the specific behaviors and outcomes of this study directly to events of developmental, physiologic, or pathologic importance. While authors do broadly invoke these as motivations for the work, the true impact of the findings is not fully realized without more direct links. Further, because the work is largely descriptive, and lacks direct measurement of cell generated forces, it does not truly take full advantage of the artificiality of the system.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    This manuscript is in revision at eLife.

    ###Summary:

    The authors study the effect of confinement on the alignment of REF cells confined within circular micropatterned islands. They observed that the cells are aligned perpendicularly to the boundary after 48h, contrary to other elongated cells such as NIH-3T3. After testing several subclones of that cell line, they identified cell contractility and cell-cell adhesion affect the organization of the cells in the circular patterns. They confirmed this finding using drugs that affect contractility and disrupt cell adhesion. Then they compared their results to a continuum model and to a Voronoi model.

    Enthusiasm for the work is diminished by the limited experimental support for key assumptions of the conceptual and math models (e.g. existence of stiffness gradient, assumption of uniform contractility, use of calcium chelator to show importance of adhesion). Further, integration of model and experiment could be improved, and some of the narrower assumptions of the models (e.g. omitting cell proliferation, remodeling of cell-cell contacts, and cell-substrate interactions, assuming uniform contractility) need better justification. Also, a clear correlation to specific events in development, physiology, or disease would highlight the broader impact of the work beyond a very specific event in a carefully engineered system. Finally, 3 similar papers came out on arxiv from the Roux group. They should be discussed in the manuscript and cited.

  7. Version published to 10.1101/2020.06.23.168237v1 on bioRxiv