Formation of adherens junctions leads to the emergence of a tissue-level tension in epithelial monolayers.
Bottom Line: Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium.Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established.As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.
Affiliation: London Centre for Nanotechnology, University College London, London WC1H 0AH, UK Department of Physics, University College London, London WC1E 6BT, UK Engineering Doctorate Program, Department of Chemistry, University College London, London WC1H 0AJ, UK.Show MeSH
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Mentions: We reasoned that by applying deep indentations onto cell monolayers growing on soft collagen gels, we should be able to mechanically deform the cell directly in contact with the cantilever tip, as well as its surrounding neighbours (Fig. 1A–E; supplementary material Fig. S1B). The slope of the acquired force–indentation curves should yield an apparent stiffness for the monolayer-gel composite (supplementary material Fig. S1C) and, if the indentation is sufficiently deep, this mechanical parameter should be sensitive to stretching of the monolayer around the location of indentation. To test this hypothesis, we used MDCK-II cells as a junction-forming epithelial cell model, culturing them on top of thick collagen gels (∼200 µm thick) with an elasticity sevenfold lower than that of the cells (Ecollagen = 66±8 Pa, Ecells≈400 Pa; ±s.d.) (Harris and Charras, 2011). We indented monolayer–gel composites using cantilevers with a cylindrical tip to obtain a constant contact area (∼80 µm2) that was smaller than the typical cellular apical area (∼300 µm2). Indentation depths of 10–15 µm, larger than the monolayer thickness, induced a deformation field that propagated over several cell diameters (Fig. 1A,B, white arrowhead; supplementary material Movie 1; Fig. S1B). Quantification of the strain field away from the location of indentation using confocal zx profiles (supplementary material Fig. S2A,B) revealed that the first and second neighbours were significantly stretched by indentation (Fig. 1C–E). Importantly, apparent stiffness, as measured by deep AFM indentation, was sensitive to the presence of intercellular adhesions. We compared the apparent stiffness of control monolayers, the collagen gel alone and monolayers in which intercellular adhesion had been disrupted by EDTA-dependent calcium chelation. Control monolayers grown on gels had an apparent stiffness that was approximately threefold greater than that of the collagen substrate alone (Fig. 1F, Kcontrol = 2.8±0.5 mN/m, Kgel = 1.0±0.1 mN/m, P<0.01; ±s.d.). Monolayers disaggregated by EDTA had a stiffness that was closer to that of the gel and significantly lower than that of control monolayers (Fig. 1F, KEDTA = 1.4±0.3 mN/m, P<0.01 when compared with control monolayers). Together with the emergence of a planar strain field in the monolayer in response to indentation (Fig. 1C–E), these data suggested that stresses induced by indentation were transmitted across intercellular junctions. We reasoned that if intercellular junctions propagate stresses, then the radial distance at which the monolayer–gel composite vertical displacement reaches zero should be larger in control monolayers than in monolayers in which intercellular adhesion was disrupted. In zx confocal images, the vertical displacement profile had a larger radius in control monolayers than in monolayers treated with EDTA (∼150 µm versus ∼90 µm to reach zero vertical displacement, n = 10 curves examined, supplementary material Fig. S2C,D). Taken together, these experiments showed that deep AFM indentation could be used to probe the mechanical properties of monolayers and that these measurements were sensitive to the presence of intercellular junctions.
Affiliation: London Centre for Nanotechnology, University College London, London WC1H 0AH, UK Department of Physics, University College London, London WC1E 6BT, UK Engineering Doctorate Program, Department of Chemistry, University College London, London WC1H 0AJ, UK.