Time-lapse DIC (left) and GFP-zyxin (right) images of a corneal fibroblast, 18 hours after plating on top of a fibrillar collagen matrix. Using DIC imaging, membrane ruffling during lamellipodial extension was clearly observed at the leading edge (upper right). Adhesions were generally oriented in a radial pattern in the front of the cell. New adhesions often formed at the leading edge while existing adhesions moved backward centripetally in a retrograde fashion and eventually disappeared. This process generated tractional forces on the ECM as indicated by pulling in of the ECM in front of the cell. At the same time, cell body and the adjacent ECM moved forward. The cycle of membrane ruffling and new adhesion formation often repeated itself as the cell continued to move forward. Localized rupture of adhesions and consequent retraction of the lower right portion of the lamellipodia was also observed near the beginning of this sequence. The time shown is relative to the start of time-lapse imaging.

Comparison of ECM displacements with focal adhesion dynamics in living corneal fibroblasts, on top of fibrillar collagen matrices. The red tracks show ECM displacements (cross indicates initial position in the sequence), and GFP-zyxin is shown in green. The adhesions at the leading edge (upper right) moved inward during cell extension. At the same time, the cell body moved forward. These movements appear to correlate with the ECM deformation. The time shown is relative to the start of time-lapse imaging.

Time-lapse color overlay images of a fibroblast in which a slow retraction of the lamellipodium was observed. The red tracks show ECM displacements (cross indicates initial position in the sequence), and GFP-zyxin is shown in green. During retraction, adhesions at the front of the cell (top) moved backward in unison and generated significant ECM deformation. At the same time, cell body adhesions moved forward, resulting in contractile-like shortening of the cell and ECM compression at the base of lamellipodia (arrows in last frame). The time shown is relative to the start of time-lapse imaging.

Time-lapse DIC images of a fibroblast in which a slow retraction of the lamellipodium was observed. During retraction, the ECM near the front of the cell (top) moved backward while the cell body moved forward, resulting in contractile-like shortening of the cell and ECM compression at the base of lamellipodia (arrows in last frame). The time shown is relative to the start of time-lapse imaging.

Time-lapse DIC (left) and GFP-zyxin (right) images of a fibroblast in which rupture of adhesions at the tail of the cell was observed. Rupture of adhesions resulted in rapid retraction of the tail, release of tension on the ECM, and forward displacement of the cell body. Interestingly, some of the adhesions are left behind (arrows on right panel), suggesting that they were “torn off” as the cell pulled forward. The time shown is relative to the start of time-lapse imaging.

DIC images demonstrating the effects of Triton X-100 on ECM deformation in GFP-zyxin-transfected living corneal fibroblasts plated on top of fibrillar collagen matrices. Prior to addition of Triton X-100, membrane ruffling and lamellipodial extension were observed at the leading edge (upper left). The ECM in front of the cell was pulled inward as the cell body moved forward. Within three minutes after adding Triton X-100 to the media, the cell rounded up and detached from the collagen ECM, and decompression of the ECM between the cell body and lamellipodium was observed (arrows in last frame). The time shown is relative to the start of time-lapse imaging.

DIC images demonstrating the effects of cytochalasin D on ECM deformation in untransfected living corneal fibroblasts plated on top of fibrillar collagen matrices. Within two minutes after adding cytochalasin D to the media, cell elongation and ECM relaxation at the front of the cell (right) was observed, resulting in decompression of the ECM between the cell body and lamellipodium (arrow in last frame). The time shown is relative to the start of time-lapse imaging.