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W Petroll, JV Jester, CJ Chuong, L Ma, CA Otey; Direct Correlation Of Extracellular Matrix Deformation With Changes In Focal Adhesion Organization In Living Corneal Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2935.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate how changes in the organization of cytoskeletal and adhesive proteins correlate with force generation by corneal fibroblasts on a fibrillar collagen extracellular matrix (ECM). Methods: Primary cultures of rabbit corneal fibroblasts (NRK) and a telomerase-infected, extended life-span human corneal fibroblast cell line (THK) were transfected using vectors for enhanced green fluorescent protein (EGFP)-zyxin (a gift from Dr. Klemens Wehland), and EGFP-α-actinin; zyxin is a component of focal adhesions and α-actinin a component of both stress fibers and focal adhesions. Cells were plated at low density on top of 100 µm thick fibrillar collagen lattices embedded with 2 µm diameter red fluorescent beads. Time-lapse imaging was performed at 1 minute intervals for up to 2 hours using a Nikon TE300 microscope. At each time interval, EGFP, PI (for beads) and DIC images were acquired in rapid succession using filter wheels. Results: Both EGFP-zyxin and EGFP-α-actinin were detected in focal adhesions of spread THK and NRK cells as confirmed using vinculin counterstaining. Time-lapse imaging 2-24 hours after plating revealed extensions and retractions of cell processes and displacements of the fiduciary beads that was similar to untransfected control cells. Tension was generated on the ECM during both extension and partial retraction of cell processes. Overall, focal adhesion movement and turnover was more dynamic than has been observed previously on rigid or planar elastic substrates. Extending processes exhibited the most complex behavior, with new focal adhesions forming at the leading edge while existing adhesions moved in a retrograde fashion. In contrast, adhesions in retracting processes appeared to pull directly on the matrix without the formation of new adhesive structures. Rupture of adhesions led to rapid and complete retraction with release of tension on the lattice. Stress fibers labeled with EGFP-α-actinin were detected 48-72 hours after plating, suggesting that stress fibers form as tension is developed within the lattice. Conclusion: Although others have studied focal adhesion dynamics on planar elastic substrates, to our knowledge this is the first such study on a fibrillar collagen matrix. Interestingly, our data suggests that isolated corneal fibroblasts may generate tension via two distinct mechanisms: traction during extension, and contraction during retraction.
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