Infoscience

Thesis

Cadherins establish mechano-resistant junctions between fibrogenic fibroblasts

During wound healing and in a variety of fibrocontractive diseases, fibroblasts differentiate into contractile myofibroblasts by acquiring stress fibers and by expressing α-smooth muscle actin (α-SMA). Myofibroblasts of wound granulation tissue, in contrast to dermal fibroblasts, join stress fibers at sites of cadherin-type intercellular adherens junctions (AJs). However, the function of myofibroblast AJs, their molecular composition, and the mechanisms of their formation are largely unknown. We demonstrate here that fibroblasts change cadherin expression from N-cadherin in early wounds to OB-cadherin in contractile wounds, populated with α-SMA-positive myofibroblasts. A similar shift occurs during myofibroblast differentiation in culture and seems to be responsible for the homotypic segregation of α-SMA-positive and -negative fibroblasts in suspension. By measuring the adhesion strength between suspended cells with a laser tweezers, we show that fibroblast adhesion is cadherin-mediated since anti-OB-cadherin and anti-N-cadherin peptides reduce the adhesion of both myofibroblasts and fibroblasts respectively. The switch from N- to OB-cadherin expression results in 1.3-fold increased adhesion strength despite reduced cadherin expression levels. Moreover, AJs of plated myofibroblasts are reinforced by α-SMA-mediated contractile activity, resulting in high mechanical resistance as demonstrated by subjecting cell pairs to hydrodynamic forces in a flow chamber. A peptide that inhibits α-SMA-mediated contractile force causes the reorganization of large stripe-like AJs to belt-like contacts as shown for enhanced green fluorescent protein-α-catenin-transfected cells and is associated with a reduced mechanical resistance. Anti-OB-cadherin but not anti-N-cadherin peptides reduce the contraction of myofibroblast-populated collagen gels, suggesting that AJs are instrumental for myofibroblast contractile activity. Using atomic force microscopy we further investigate the difference between N- and OB-cadherin adhesion strength on the single molecule level and show that part of the stronger adhesion of myofibroblasts is accomplished because single OB-cadherin bonds resist 2-fold higher forces compared with single N-cadherin junctions. By assessing the adhesion force between recombinant cadherin dimers and between native cadherins in the membrane of spread fibroblasts, we demonstrate that cadherin bonds are reinforced over time, displaying a hierarchy of three distinct adhesion forces. By modulating the degree of lateral cadherin diffusion and of F-actin organization we can attribute this resulting three force states to the single molecule bond rather than to cadherin cluster formation; this is consistent with a new model of homotypic cadherin ectodomain interaction. Notably, association with actin filaments enhances cadherin adhesion strength on the single molecule level by up to 3-fold; actin depolymerization reduces single bond strength to the level of cadherin constructs missing the cytoplasmic domain. Hence, fibroblasts reinforce intercellular contacts by: 1) switching from N- to OB-cadherin expression, 2) increasing the strength of single molecule bonds in three distinct steps, and 3) actin-promoted intrinsic activation of cadherin extracellular binding. We propose that this plasticity adapts fibroblast adhesions to the changing mechanical microenvironment of tissue under remodeling.

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