Abstract
Maintenance of epithelial tissue integrity requires coordination between cell–cell adherens junctions, tight junctions (TJ), and the perijunctional actomyosin cytoskeleton. Here we addressed the hypothesis that alterations in TJ structure and remodeling of the actomyosin cytoskeleton modify epithelial mechanics. Current methods to measure supracellular mechanical properties disrupt intact monolayers, therefore, we developed a novel method using noncontact acoustic frequency-modulation atomic force microscopy (FM-AFM) and tested it on MDCK polarized monolayers. Our results show that double knockdown (dKD) of ZO-1/ZO-2 elevates the apical epithelial tension and effective viscosity. Interestingly, epithelial tension is more sensitive to inhibition of myosin II ATPase activity than to inhibition of ROCK activity, but viscosity is highly sensitive to both. Additionally, we showed epithelial intercellular pulling forces at tricellular junctions and adhesion forces in dKD cells are elevated with an increase in contractility. In conclusion, FM-AFM enables the physiological and quantitative investigation of mechanics in intact epithelium.
Highlights
Maintenance of epithelial tissue integrity requires coordination between cell–cell adherens junctions, tight junctions (TJ), and the perijunctional actomyosin cytoskeleton
Mature cell–cell adhesions have fully developed adherens junctions, with transmembrane cadherin proteins linked through catenins to an ultrastructurally distinct highly contractile actomyosin belt and tight junctions, with transmembrane adhesion proteins claudins, occludin, and JAMAs linked through ZO proteins to a less distinct actin cytoskeleton plaque (Fig. 1b)
We demonstrated that several supracellular mechanical properties - epithelial tension, effective viscosity, intercellular forces pulling on the tricellular junctions, and intercellular adhesion forces - can be extracted on polarized monolayers by a noninvasive Atomic force microscopy (AFM) method compatible with any commercially available AFM system
Summary
Maintenance of epithelial tissue integrity requires coordination between cell–cell adherens junctions, tight junctions (TJ), and the perijunctional actomyosin cytoskeleton. It is thought that AJ tension-sensitive regulation of the actomyosin cytoskeleton may help reinforce cell–cell adhesions against increased intercellular forces in epithelial monolayers[7, 8]. The depletion of ZO-1 and/or ZO-2 has been shown to cause dramatic reorganization of the perijunctional actomyosin cytoskeleton into large thick arrays of F-actin under the adherens junction contacts and recruitment of myosin II bipolar minifilaments, forming a contractile sarcomeric belt-like distribution[11]. Using laser ablation to cut cell–cell contacts, it was shown that elevated junctional contractility in ZO KD cells results in a dramatic increase in recoil velocity, implying an elevated epithelial tension[12]. Laser ablation is a disruptive technique, and cutting the cell–cell contacts of adjacent cells makes it difficult to determine any specific contribution of alterations of tight junction structure to epithelial tension and intercellular forces. Quantitative determination of critical physical parameters is key for understanding how forces are transmitted within and between cells during important cellular processes and for providing insight into regulation of these processes
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