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: In deep AFM indentation, monolayer apparent stiffness results from a combination of intercellular tension and traction stresses. We focussed our attention on the contribution of intercellular junctions. To investigate the role of adherens junctions and desmosomes, we disrupted the function of E-cadherin and desmoplakin. First, we disrupted E-cadherin-mediated adhesion with a blocking antibody that binds to the ectodomain of E-cadherin (Gumbiner et al., 1988) (Fig. 4A). Under these conditions, cells were able to spread onto the collagen substrate but E-cadherin no longer localised to the cell membrane, and the cells were unable to form intercellular junctions (Fig. 4A). Consistent with our hypothesis, this treatment significantly reduced the increase in apparent stiffness observed following replating (Fig. 4B). Interestingly, in the presence of E-cadherin-blocking antibody, apparent stiffness remained significantly larger than that of the collagen gel, indicating that deep AFM indentation is sensitive to traction stresses. A comparison with control monolayers suggested that traction stresses accounted for at most 50% of the measured increase in apparent stiffness (Fig. 4B). Second, we disrupted desmosomes by generating a cell line stably expressing the N-terminal portion of desmoplakin (DPNTP) tagged with GFP. DPNTP binds to the desmosomal plaque but lacks a keratin-binding domain and, when overexpressed, acts as a dominant negative mutant that prevents the association of the intermediate filament network with desmosomes (Bornslaeger et al., 1996; Huen et al., 2002). Consistent with previous reports, overexpression of DPNTP displaced endogenous desmoplakin from intercellular junctions (compare Fig. 4C with Fig. 3C) and caused keratin intermediate filaments to concentrate in the perinuclear area in mature monolayers [compare Fig. 4D with Fig. 3D (Huen et al., 2002)]. As an intercellular keratin filament network that interfaced with desmosomes formed between 5 h and 18 h after plating (Fig. 3C,D), we reasoned that the impact of desmosome perturbation on monolayer stiffness should be most apparent in mature monolayers plated for 18 h. However, despite the dramatic changes in desmosomal organisation that were induced by DPNTP overexpression, the apparent stiffness of mature DPNTP monolayers was not significantly affected (Fig. 4E, KControl = 2.7 mN/m, n = 60; KDPNTP = 2.9 mN/m, n = 39; P = 0.08). Collectively, these data show that the assembly of adherens junctions coincides with the establishment of a tissue-level tension during monolayer formation and that desmosomes do not play a role in the establishment of tissue tension.
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.