Analysis of the pattern of subcellular force generation by corneal fibroblasts after Rho activation

PURPOSE. To determine the structural and subcellular mechanical effects of Rho activation on corneal fibroblasts in three-dimensional collagen matrices. METHODS. Human corneal fibroblasts were plated at low density in 100-μm thick fibrillar collagen matrices and cultured for 1 or 2 days in serum-free media. Time-lapse imaging was then performed at 1- to 2-minute intervals with Nomarski differential interference contrast. After 1 hour, perfusion was switched to serum-free media containing 1 μmol/L lysophosphatidic acid (LPA). After an additional 30 to 60 minutes, the Rho kinase (ROCK) inhibitor Y-27632 was added to the perfusion media. Changes in cell structure and extracellular matrix deformation were measured with MetaMorph. RESULTS. Addition of LPA activated Rho and induced retraction of cell processes and cellular contraction, as indicated by decreases in cell length (-12.1% ± 7.0%; P<0.05) and cell area (-13.1% ± 13.5%; P=0.06). Force generation was greatest along the cell body in all cases, as indicated by the location of maximum extracellular matrix compression. Subsequent addition of Y-27632 resulted in relaxation of extracellular matrix stress, and reextension of cellular processes. CONCLUSIONS. The data show that Rho induces rapid contraction of corneal fibroblasts in three-dimensional collagen matrices. Forces are generated primarily along the cell body through a ROCK-dependent mechanism.