Direct correlation of collagen matrix deformation with focal adhesion dynamics in living corneal fibroblasts

Abstract

The purpose of this study was to develop and apply a new model for investigating how the organization and movement of cell-matrix adhesion sites correlate with force generation by corneal fibroblasts on a fibrillar collagen extracellular matrix. Primary cultures of rabbit corneal fibroblasts were transfected using a vector encoding GFP-zyxin to allow visualization of adhesion sites. 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 one minute intervals for up to 3 hours. At each time interval, GFP-zyxin, bead and DIC images were acquired in rapid succession using filter wheels. Cells were treated with cytochalasin D and/or Triton X-100 at the end of each experiment. The movements of adhesions and nearby matrix landmarks were measured and correlated from the time-lapse digital images, and the size, intensity and orientation of the adhesions were quantified.GFP-zyxin was detected in adhesions of transfected corneal fibroblasts as confirmed using vinculin counterstaining. Time-lapse imaging revealed extensions and retractions of cell processes and displacements of the fiduciary beads that were similar to control cells. Extending processes exhibited the most complex behavior, with new adhesions continuously forming at the leading edge while existing adhesions moved backward in a retrograde fashion. This process generated tractional forces as indicated by pulling in of the extracellular matrix in front of the cell. Interestingly, during extension, adhesions along the ventral surface of the cell body generally moved toward those at the tip, resulting in contractile-like shortening and matrix compression at the base of lamellipodia. Overall, a high correlation was found between both the magnitude (R=0.87, P<0.001) and direction (R=0.98, P<0.001) of the adhesions and nearby matrix displacements. Cytochalasin D induced rapid and reversible disassembly of adhesions, cell elongation and matrix relaxation, including decompression at the base of the lamellipodia. This new experimental model allows direct,dynamic assessment of cell-matrix interactions on a fibrillar collagen matrix. Our results are consistent with the previously described `frontal towing'model of cell motility and demonstrate for the first time that this mechanism is employed by cells interacting with a fibrillar extracellular matrix.