Abstract
Abstract: :
Purpose: To investigate the response of corneal fibroblasts to dynamic changes in extracellular matrix (ECM) tension. Methods: Rabbit and human corneal fibroblasts were plated at low density inside 100 µm thick type I collagen matrices and allowed to spread for 24 – 48 hours. Following 1 hour of time–lapse DIC imaging, a glass microneedle was inserted into the ECM approximately 75 µm from the leading edge of a cell of interest. The needle was either pushed toward the cell to reduce ECM tension (29 cells), or pulled away from the cell to increase ECM tension (9 cells). Time–lapse imaging was then continued for an additional 2–3 hours. Dynamic changes in focal adhesion organization were evaluated in a subset of cells transfected to express GFP–zyxin, by using simultaneous fluorescent and DIC imaging. In some experiments, cells were treated with the Rho–kinase inhibitor Y–27632 either 30 minutes prior to, or 1 hour after ECM micromanipulation. The effect of PDGF was also evaluated in 7 cells without micromanipulation. ECM displacements were tracked using MetaMorph, and maps of mechanical stress and strain were generated using finite element modeling. Results: Pulling on the ECM resulted in increased tension and cell elongation. Following the initial stretch, pseudopodia often disengaged and retracted (thus reducing cellular tension); cells then re–spread without significantly displacing the ECM. In contrast, reducing ECM tension by pushing the needle toward a cell induced an immediate rapid cellular contraction, presumably since existing cellular forces were no longer counterbalanced by the ECM. ECM displacement was correlated with the inward movement of existing focal adhesions toward the cell body. Following this initial contraction, pseudopodial extension was observed at both ends of the cell, and additional tractional force was generated as indicated by gradual restoration of baseline ECM tension. Traction was associated with formation of new focal adhesions at pseudopodial tips, and rearward movement of existing adhesions in a retrograde pattern. Cell spreading and tractional force generation could also be induced by treating unmanipulated cells with PDGF, which upregulates Rac. Addition of Y–27632 following the needle push resulted in rapid cell elongation and loss of cell–induced ECM tension, and pre–incubation with Y–27632 completely blocked the cellular response to ECM manipulation. Conclusions: The data suggest that corneal fibroblasts actively respond to increases or decreases in local matrix stress in an attempt to maintain tensional homeostasis (constant tension), and that this response is mediated by both Rho and Rac.
Keywords: cornea: stroma and keratocytes • wound healing • microscopy: light/fluorescence/immunohistochemistry