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Apico-basal cell compression regulates Lamin A/C levels in Epithelial tissues

K Venkatesan Iyer, Natalie A. Dye, Suzanne Eaton, Frank Jülicher

Preprint posted on May 19, 2020 https://www.biorxiv.org/content/10.1101/2020.05.18.102509v1

An evolutionary conserved mechanism in epithelia dictates Lamin A/C levels via nuclear deformation

Selected by Ilaria Di Meglio

Categories: biophysics, cell biology

BACKGROUND

Physical cues and forces are ever-present in native tissues; tissue properties like substrate stiffness or cues like compressive stress generated by increasing cell density can dictate biological function and cell behaviour. The process by which cells transduce physical forces from the microenvironment into an intracellular biochemical response is termed mechanotransduction. The field of mechanobiology has grown enormously in the last decades, revealing an increasing number of molecular players that relay mechanical forces to signalling pathways that ultimately dictate gene expression. The nucleus has emerged as one of these players, providing an important platform for mechanotransduction [1]. For instance, increased actomyosin contractility on stiff substrates can deform the nucleus to regulate differentiation [2] and proliferation [3]. The mechanical properties of the nucleus, which are defined primarily by the nuclear lamina, play an important role in nuclear mechanosensation. The nuclear lamina consists primarily of intermediate filament proteins A-type and B-type lamins; in vertebrates, the A-type lamin isoforms are Lamin A and C (lamin C in invertebrates) and the B-type lamins are lamin B1 and B2 (lamin Dm0 in invertebrates). Recently, Lamin A/C has received particular attention because it confers stiffness to the nucleus, and in tissues like the mesenchyme Lamin A/C is actually regulated by substrate stiffness [2]. However, the role of Lamin A/C in epithelial tissues, where cell-cell adhesion (rather than cell-ECM adhesion) bears most of the mechanical stress, is less clear. With this in mind, Iyer et al. assess the role of Lamin A/C in two epithelial model systems, Drosophila epithelial tissues and an in vitro MDCK monolayer.

Key findings

With the aim of understanding Lamin A/C regulation in epithelial tissues, the authors first assessed Lamin A/C levels via immunofluorescence in different Drosophila epithelial tissues; the salivary gland (SG), the trachea and the wing disc. Lamin A/C is in fact differentially expressed amongst these tissues, with lowest levels in the salivary gland and highest in the trachea. Within the wing disc, Lamin A/C levels are highest in the Peripodial Membrane (PM) cells of the wing, decreasing in the folds and lowest in the wing pouch (Fig 1a).

Naturally, the authors next sought to better understand what underlies this regulation, focusing first on cell packing density. To investigate how cell packing or apico-basal compression impacts Lamin C, the authors quantify the cell deformation index (Dcell) by measuring apical cell area (A) and apico-basal height (h). In this way, cuboidal cells have an index of approx.  0, columnar cells <0 and squamous cells >0 (Fig 1b). By comparing Dcell index in the different tissues and plotting Lamin C concentration as a function of Dcell, the authors find that Lamin C does scale with apico-basal compression (Fig 1c).

Figure 1: Lamin C levels depend on epithelial apico-basal compression. (a) Epithelial tissues in Drosophila express different levels of Lamin C. (b) Quantification of the cell deformation index Dcell to assess cell morphology reveals that (c) Lamin C correlates linearly with apico-basal deformation (right). Figure adapted from Iyer et al.

 

Based on previous findings for Lamin C regulation in mesenchymal cells, the authors assess whether Lamin C regulation also depends on ECM concentration in epithelial cells by (i) quantifying collagen IV levels and (ii) depleting ECM in the wing pouch using Dpp-GAL4 to express matrix metalloprotease (MMP2), known to induce ectopic folds. However, the authors find that Lamin C concentration does not correlate with collagen IV levels, and ECM depletion does not affect Lamin C levels between the wing pouch and ectopic folds. Since ECM concentration does not regulate Lamin C levels in Drosophila epithelial tissues, the authors investigate whether apico-basal compression does. By tuning cell shape using a dominant negative mutant of CDC42 (CDC42F89) to reduce apico-basal height and overexpressing the Lines transcription factor to increase apico-basal height, the authors find the same linear correlation between Dcell and normalized Lamin C levels as in wild-type tissue. The apico-basal deformation of epithelial cells can therefore be used as a direct predictor of Lamin A/C levels. Interestingly, while apico-basal deformation regulates Lamin A/C levels, the reverse does not occur. In fact, by changing Lamin C expression levels, the authors find no significant differences in apico-basal cell height. This indicates there is a unidirectional coupling wherein apico-basal compression regulates Lamin C but Lamin C does not regulate cell shape. At this stage, the authors ask whether nuclear deformation is at the heart of Lamin A/C regulation by apico-basal compression. As with cell deformation index, the authors quantify a nuclear deformation index (Dnuc) and compare nuclear deformation in the salivary glands, trachea and wing disc. Interestingly, Dnuc strongly correlates with Dcell, which indicates that apico-basal compression is in fact sensed through deformation of the nucleus to dictate Lamin C levels.

Finally, the authors investigate whether the regulation of Lamin A/C found in Drosophila epithelial tissues also occurs in mammalian tissues. By culturing MDCK cells at different cell densities, Iyer et al., find that nuclear deformation in MDCK also strongly correlates with cell deformation, as does the concentration and total amount of Lamin A. Therefore, Lamin A/C regulation by apico-basal compression is conserved from Drosophila to mammalian cells.

What I liked about this preprint

I truly enjoyed reading this preprint, it was both extremely interesting and insightful. The role of the nucleus as a mechanosensor is increasingly appreciated, but the underlying mechanisms by which this regulation occurs are not clear – this study brings us a step closer to understanding this regulation. What I found particularly interesting and convincing was the comparison of epithelial tissues within the same model organism, but also the use of both invertebrates and vertebrates to show how the regulation of Lamin A/C via cell morphology is actually conserved in epithelial tissues.

Questions and future directions

  1. The notion that size determination is regulated by mechanical feedback has been proposed extensively, particularly for development of the Drosophila wing disc, but the underlying mechanisms have not been fully unravelled. Could the regulation of Lamin A/C via cell packing be involved? Have you quantified Lamin C as the wing grows to compare levels in the centre and periphery of the wing pouch?
  2. The authors investigate Lamin A/C regulation in MDCK tissues cultured on collagen I coated glass dishes in what they say is an ECM independent environment. How can you dismiss the effect of the stiffness of the glass on Lamin A/C levels? Could you tune substrate stiffness to ensure that it does not contribute to Lamin A/C regulation?

References

  1. Kirby, T.J. and J. Lammerding, Emerging views of the nucleus as a cellular mechanosensor. Nat Cell Biol, 2018. 20(4): p. 373-381.
  2. Swift, J., et al., Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science, 2013. 341(6149): p. 1240104.
  3. Elosegui-Artola, A., et al., Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores. Cell, 2017. 171(6): p. 1397-1410 e14.

Tags: drosophila, epithelia, mdck, mechanical stress, nuclear mechanics

Posted on: 5th August 2020 , updated on: 10th August 2020

doi: https://doi.org/10.1242/prelights.23733

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  • Author's response

    K Venkatesan Iyer shared

    Dear Ilaria,

    Thanks for the elaborate and nice summary of our work and insightful questions. Here are our responses to your questions:

    1. The notion that size determination is regulated by mechanical feedback has been proposed extensively, particularly for development of the Drosophila wing disc, but the underlying mechanisms have not been fully unravelled. Could the regulation of Lamin A/C via cell packing be involved? Have you quantified Lamin C as the wing grows to compare levels in the centre and periphery of the wing pouch?

    It would be interesting to explore this idea that regulation of levels of Lamin A/C could be the underlying mechanism of the mechanical feedback between cell packing and wing size. Measuring the levels of Lamin C spatially in Drosophila wing disc during development would be very informative to address this question. But the existing protein trap lines available do not have a very strong GFP expression of Lamin C in the wing disc. Generating a CRISPR mediated endogenously tagged LaminC GFP fly line would the best approach. Though it is beyond the scope of the current work, we have plans of pursuing this in our future work.

    1. The authors investigate Lamin A/C regulation in MDCK tissues cultured on collagen I coated glass dishes in what they say is an ECM independent environment. How can you dismiss the effect of the stiffness of the glass on Lamin A/C levels? Could you tune substrate stiffness to ensure that it does not contribute to Lamin A/C regulation?

    It is an interesting question. In our work we are not dismissing the effect of stiffness of glass on Lamin A/C levels. In our experiments we have cultured cells in low and high density on cover glass coated with same concentration of collagen. This ensures that cells in low and high density cultures are exposed to the same concentration of collagen and the same stiffness of glass. The only difference between these cultures is the packing of cells. Thus, the design of the experiment makes Lamin A/C levels ECM and stiffness independent. The idea of tuning the ECM stiffness is interesting. This is an ongoing work in the lab to grow these cells on PDMS gels of varying stiffness and compare the Lamin A levels in the low density and high density cultures.

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