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Large-scale curvature sensing by epithelial monolayers depends on active cell mechanics and nuclear mechanoadaptation

Marine Luciano, Shi-Lei Xue, Winnok H. De Vos, Lorena Redondo Morata, Mathieu Surin, Frank Lafont, Edouard Hannezo, Sylvain Gabriele

Preprint posted on July 04, 2020 https://www.biorxiv.org/content/10.1101/2020.07.04.187468v1

Sensing the curve: how epithelial cells modulate morphology and nuclear activity to adapt to curvature

Selected by Grace Lim, Ilaria Di Meglio

Categories: biophysics, cell biology

Background

In native tissues, curvature is found everywhere. During tissue morphogenesis, cellular sheets deform via invaginations, budding and cavitation, amongst other processes, to generate 3D shapes like tubes and cysts of the lungs or kidneys, or the concave and convex curvature that characterize the crypts and the villi of the intestine. Curvature contributes structurally to most tissues, often by providing the increased surface area necessary for absorption like in the intestine, or gas exchange in the lungs. It is becoming increasingly clear that curvature also serves a functional purpose; curvature contributes to cell migration [1], to cell fate specification [2], and even to disease progression [3]. However, the underlying mechanisms by which cells sense local curvature and how it ultimately dictates cell behavior is less clear. In this study, Luciano et al. produce corrugated hydrogels with isotropic wavy patterns to investigate the effect of convex and concave curvatures on epithelial cells and nuclear shape.

Key findings

To understand how curvature of epithelial tissues can influence their functions, the authors first engineered corrugated hydrogels with well-defined curvature profiles. This was achieved using an optical photomask with alternating transparent and black stripes together with a photopolymerization method initiated by UV light (Fig. 1). Varying the width of the alternating stripes in the photomask enabled the generation of two types of corrugated substrates utilised in this study: P20 and P30, with sinusoidal patterns having 20mm and 30mm wavelengths respectively (Fig. 1). This approach enabled the authors to precisely control the wavelength and amplitude of the curved patterns, giving them a reliable system to answer their research questions.

Figure 1. Generation of corrugated hydrogels. A photomask with alternating transparent and black stripes was used to produce patterns of UV light penetration to activate a photopolymerization reaction (left). Measurements of wavelength and amplitude from the P20 and P30 hydrogels (right). Adapted from Figure 1 of Luciano et al.

Epithelial cells grown on these corrugated substrates retained their overall epithelial morphology and cytoskeletal organization, maintaining their cell-cell interactions and characteristic hexagonal arrays. In contrast, cells exhibited differences in shape on flat vs. curved substrates. Cells on crests (concave) and valleys (convex) exhibited a mean 20% difference in thickness, wherein cells on crests extended along the tissue plane (squamous-like aspect ratio) while cells on valleys extended along the apico-basal direction (columnar-like aspect ratio). These findings suggest that curvature may be sensed by cells in an indirect manner by creating differences in thickness/density. To assess how curvature could create such cell shape modulations, Luciano et al., turn to a theoretical model.

The theoretical model used in this study is based on the vertex model, which has been used extensively to investigate the mechanisms and mechanics underlying epithelial tissue deformations [4]. Briefly, the vertex model describes a force balance equation that depends on three major contributions to the effective energy: a cell-substrate energy (proportional to the basal area), a cell-cell energy (proportional to the lateral area) and an energy coming from actomyosin-based tension (proportional to the apical perimeter). The authors adapt this model to their system by assuming that the basal area cannot detach from the substrate such as to remove the contribution of cell-substrate energy. Using numerical simulations to tune multiple parameters like wavelength, amplitude and tensions, and fitting these to experimental data, the analytical theory suggests that changes in substrate curvature into changes in cell thickness/density can be explained via a purely physical mechanism involving active contractile apico-basal tensions.

In addition to changes in cell shape, epithelial cells grown on corrugated substrates also display different nuclear morphologies, localization, and orientation. The nuclei of cells located in concave areas (crests) displayed an oblate shape, whereas those in convex regions (valleys) adopted a prolate shape – nuclear morphologies that can be attributed to changes in cell shape by an extension of the vertex model. Moreover, the authors found a bias in nuclear localization towards valleys where cell thickness is highest, and these nuclei also oriented more closely along the corrugation axis, as compared to nuclei of cells located in concave crest regions. Their theoretical model suggests that this could be an energy minimization step to reduce nuclear deformation resulting from cell shape changes and substrate curvature.

Based on the finding that substrate curvature modulates nuclear morphology, the authors next sought to investigate whether nuclear mechanics translates to changes in YAP localization. Recently, YAP localization was shown to depend on nuclear deformations. For instance, increased substrate stiffness physically deforms the nucleus and regulates the transport of YAP through nuclear pores [5], while circumferential actomyosin belt contraction induced by high cell density can drive nuclear export of YAP [6]. YAP may therefore sense curvature to induce the observed density changes. Indeed, YAP nuclear/cytoplasmic ratio was high on the side and top of P20 and P30 curved substrates, where cell density is low, whereas it was low on flat and bottom zones, where density is high. YAP therefore localizes preferentially to positively curved surfaces, where cell density is lower, suggesting that YAP-curvature sensing could occur indirectly from density-sensing. If this hypothesis is correct, then high cell densities – which increase the contributions of lateral tensions over apical tensions – should abrogate thickness modulations. Indeed, very dense epithelial tissues exhibit low YAP nuclear/cytoplasmic ratio, independently of substrate curvature. This confirmed that YAP curvature-sensing is inhibited at high cell density.

Given the well-known mechanosensing role of the nucleus [7], the authors went on to investigate a possible alteration in the nuclear lamina resulting from changes in nuclear morphology due to substrate curvature. Interestingly, the ratio of lamin A to lamin B differed depending on substrate curvature, with nuclei experiencing positive substrate curvature displaying lower lamin A levels and those experiencing negative substrate curvature with higher lamin B levels. The varying proportion of lamins A and B has been linked to elastic and viscous mechanical properties of the nucleus, which could be a mechanosensitive response of the cell to substrate curvature.

Finally, the authors sought to investigate whether curvature-induced modulation of nuclear shape, YAP and lamin A/B levels also affects DNA synthesis and proliferation. Nuclear volumes were lowest on the bottom of both P20 and P30 corrugated hydrogels, which suggests that negative/concave curvature, where density is highest, induces nuclear shape remodeling. By measuring chromatin condensation, which can be deduced from the nucleus (DAPI) intensity to nucleus volume ratio, the authors also found that chromatin condensation was associated with the nuclear deformation observed on the bottom of P20 and P30 zones. Chromatin compaction correlated with lower rates of proliferation observed in valleys, suggesting that negative curvatures induce chromatin condensation that in turn decreases DNA synthesis. Altogether, these results suggest that nuclear deformation observed on concave/negative curvature, which is associated with high cell densities, induce chromatin compaction and inhibit cell proliferation.

Why we like this preprint

It has remained difficult to study epithelial curvature without a robust system to generate well-defined curved substrate patterns for in vitro epithelial cell culture. Here, the authors engineer a way to precisely modulate substrate curvature to reliably address this question. This was combined with careful theoretical modelling, quantitative analysis of cell and nuclei morphologies, and the identification of key molecular players including YAP and lamins to establish an important link between curvature and mechanosensing pathways in epithelial tissues. We believe that this work presents important insights that can inform our understanding of many cellular and developmental contexts, given the pervasiveness of tissue curvature in a wide number of systems.

Questions to authors

  1. The authors suggest that curvature differences could be indirectly sensed via differences in cell density – cells in concave zones displayed greater cell areas, whereas convex zones were more densely packed. Is there a way to distinguish between changes in cell density induced by curvature versus that induced by other means, such as physical confinement of cells?
  2. The authors report curvature-induced changes in lamin levels and chromatin organization as separate observations. However, given that lamins and chromatin are known to closely interact, could the authors clarify whether changes in lamin levels can directly influence chromatin organization in their system?
  3. The authors propose that YAP curvature-sensing arises indirectly from cell density differences found between concave and convex curvatures. Have the authors investigated what may lie upstream of YAP regulation; is it nuclear deformation that physically promotes YAP nuclear exclusion, or are specific proteins (such as Merlin released from AJs) involved?
  4. The authors use “soft” corrugated hydrogels of stiffness 250kPa, and while this value is several orders of magnitude softer than glass/plastic culture dishes, it is higher than most soft tissues which have elastic moduli from 100Pa to 100kPa. The kidney, for instance, is >10kPa. Could a more physiological substrate stiffness affect the sensitivity of MDCK cells to thickness/density changes induced by curvature? Can the authors tune the stiffness of the corrugated hydrogels to investigate this ?

References

  1. Yevick, H.G., et al., Architecture and migration of an epithelium on a cylindrical wire. Proc Natl Acad Sci U S A, 2015. 112(19): p. 5944-9.
  2. Mobasseri, S.A., et al., Patterning of human epidermal stem cells on undulating elastomer substrates reflects differences in cell stiffness. Acta Biomater, 2019. 87: p. 256-264.
  3. Messal, H.A., et al., Tissue curvature and apicobasal mechanical tension imbalance instruct cancer morphogenesis. Nature, 2019. 566(7742): p. 126-130.
  4. Hannezo, E., J. Prost, and J.F. Joanny, Theory of epithelial sheet morphology in three dimensions. Proc Natl Acad Sci U S A, 2014. 111(1): p. 27-32.
  5. Elosegui-Artola, A., et al., Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores. Cell, 2017. 171(6): p. 1397-1410 e14.
  6. Furukawa, K.T., et al., The Epithelial Circumferential Actin Belt Regulates YAP/TAZ through Nucleocytoplasmic Shuttling of Merlin. Cell Rep, 2017. 20(6): p. 1435-1447.
  7. Kirby, T.J. and J. Lammerding, Emerging views of the nucleus as a cellular mechanosensor. Nat Cell Biol, 2018. 20(4): p. 373-381.

 

Posted on: 10th August 2020

doi: https://doi.org/10.1242/prelights.23824

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  • Author's response

    Edouard Hannezo, Sylvain Gabriele shared

    1/ The authors suggest that curvature differences could be indirectly sensed via differences in cell density – cells in concave zones displayed greater cell areas, whereas convex zones were more densely packed. Is there a way to distinguish between changes in cell density induced by curvature versus that induced by other means, such as physical confinement of cells?

    This is a great question, and one of the things we’re exploring further now! One idea that we want to try out is to test more quantitatively how the YAP localization as a function of density on flat substrate compares to the YAP localization as a function of density on curved substrate (with different seeding densities). Our results so far show that the previously observed relationship between (flat) density/confinement and YAP localization is sufficient to explain curvature sensing, but this type of experiment would help to make this even more quantitative (or precisely quantify residual effects of curvature which cannot be explained solely by this mechanism).

    2/ The authors report curvature-induced changes in lamin levels and chromatin organization as separate observations. However, given that lamins and chromatin are known to closely interact, could the authors clarify whether changes in lamin levels can directly influence chromatin organization in their system?

    It is well established that lamins interact with chromatin either directly or indirectly through chromatin binding proteins. Among the different types of lamins, B type lamins interact with heterochromatin through the heterochromatin protein 1 alpha (HP1α), while A type lamins interact with proteins associated with both hetero and euchromatin, such as LAP2α, Emerin and BANF1. In this context, modulating the level of lamin expression is an interesting suggestion to determine a direct influence on chromatin condensation in wavy epithelial monolayers. However, this experimental approach might be hard to interpret, and we’re still thinking how to get around this. Indeed lamin depletion will also have significant effects on the mechanical properties of the nuclear envelope, that in turn will affect nuclear deformation and thus chromatin condensation via alternative potential routes. One must note also that lamins do not only localize at the lamina but also have an intranuclear pool that organizes the genome and which will also become depleted upon knockdown. An alternative way could be to use chemical compounds such as saquinavir to force prelamin A accumulation (M. Versaevel et al. Cell Adh Migr 11, 98-109 2017) and see whether this affect nuclear deformation and chromatin organization.

    3/ The authors propose that YAP curvature-sensing arises indirectly from cell density differences found between concave and convex curvatures. Have the authors investigated what may lie upstream of YAP regulation; is it nuclear deformation that physically promotes YAP nuclear exclusion, or are specific proteins (such as Merlin released from AJs) involved?

    Given past reports of forces triggering nuclear YAP entry via nuclear deformation (Elosegui et al, Cell, 2017), we think that the former is an intuitive hypothesis indeed! One issue is that to rigorously exclude other hypotheses though, we would have to get density-changes without accompanying nuclear deformations, which is technically challenging. But testing alternative pathways such as Merlin would definitely be a cool next step!

    4/ The authors use “soft” corrugated hydrogels of stiffness 250kPa, and while this value is several orders of magnitude softer than glass/plastic culture dishes, it is higher than most soft tissues which have elastic moduli from 100Pa to 100kPa. The kidney, for instance, is >10kPa. Could a more physiological substrate stiffness affect the sensitivity of MDCK cells to thickness/density changes induced by curvature? Can the authors tune the stiffness of the corrugated hydrogels to investigate this ?

    This is a very interesting suggestion. We used hydroxy-PAAm hydrogels to culture epithelial cells on softer matrices that classic glass/plastic culture dishes. Karimi and Shojaei have reported an elastic modulus for the human kidney of 180 ± 11 kPa and 95 ± 9 kPa under the axial and transversal loadings, respectively (A. Karimi and A. Shojaei IRBM 38, 292-297 2017). The kidney showed a significantly higher elastic modulus under the axial loading compared to the transverse one. We have selected a Young’s modulus of 250 kPa, which is close to the physiological situation and corresponds to the optimal window of elasticity required for vinculin assembly, actin fiber formation and, subsequently, to initiation of replication in kidney epithelial cells (L. Kocgozlu et al. J Cell Sci 123, 29-39 2010). As suggested, we could change stiffness of corrugated hydrogels towards lower Young modulus values (~1-10 kPa) by changing the acrylamide/bisacrylamide ratio or illumination parameters (exposition time or illumination power), and exploring the resulting effects would be nice indeed. Tuning the hydrogel stiffness can be therefore very useful for mimicking the physiological range of mechanical properties of softer epithelial tissues that present in vivo corrugations.

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