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First published online 25 February 2003
doi: 10.1242/jcs.00357

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

Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75390-9057, USA

View larger version (55K): [in a new window]   Fig. 1. Corneal fibroblasts transfected to express GFP-zyxin were plated on glass dishes and labeled for (A) vinculin and (B) F-actin organization. (A) GFP-zyxin (green) and vinculin (red) were co-localized into punctate spots (yellow) in the lamellipodia, as well as larger, more elongated structures along the cell body. (B) Yellow spots demonstrate that GFP-zyxin (green) is localized to the ends of the F-actin stress fibers (red), which is the expected localization of focal adhesions. Weaker GFP-zyxin labeling in a periodic pattern along the stress fibers is also observed. Bar, 20 µm.

View larger version (53K): [in a new window]   Fig. 2. DIC and fluorescent color overlay images of living corneal fibroblast expressing GFP-zyxin, 18 hours after plating on top of a fibrillar collagen matrix. (A) DIC imaging allowed detailed visualization of the fibrillar collagen organization in addition to the beads. (B) GFP-zyxin (green) was organized into adhesions that were most concentrated at the leading edge (upper right) and tail of the cell. Adhesions at the leading edge were generally organized into a radial pattern. Diffuse background GFP-zyxin labeling was also observed. Fiduciary beads are also visible (red). Bar, 20 µm. See also movie 1 at jcs.biologist.org/supplemental.

View larger version (69K): [in a new window]   Fig. 3. Investigation into whether the ECM deformation could be measured directly from DIC images. (A) A segmented DIC image. Above-threshold (red) spots were used as ECM landmarks and tracked over time. (B) Color overlays of bead (red) and DIC (green) tracks (cross indicates initial position in the sequence). Tracks that are close to each other show the same pattern of movement (circle). (C,D) The magnitude and direction of selected bead and DIC tracks taken from five experiments were analyzed. A high correlation was found between both the (C) magnitude and (D) direction of the bead and DIC displacements. Bar, 20 µm.

View larger version (120K): [in a new window]   Fig. 4. Comparison of ECM displacements with focal adhesion dynamics in living corneal fibroblasts on top of fibrillar collagen matrices. The red tracks show ECM displacements, whereas the white tracks show adhesion displacements (cross indicates initial position in the sequence). GFP-zyxin images are shown in green. The time (t) is relative to the start of time-lapse imaging. (A-D) The front of a cell moving in a northeast direction. Tracking of the adhesions confirmed our qualitative observation that the adhesions moved inward during cell extension (A-C, arrowheads), and that these movements generally correlated with the ECM deformation in front of the cell (D). See also movie 2 at jcs.biologist.org/supplemental. (E-H) A different cell moving in the northeast direction. Adhesions generally moved perpendicular to the local cell contour, i.e. in a centripetal direction (H), thereby pulling in the ECM in a radial pattern around the front of the cell. Note that as the ECM in front of the cell was pulled backward during extension, the cell body and adjacent ECM was pulled forward, resulting in ECM compression at the base of the lamellipodia. Cell body adhesions (arrows) also moved toward those at the tip (arrowheads), which is consistent with the cell body and ECM movement in this region. (I-L) A cell in which a slow retraction of the lamellipodium was observed. During retraction, adhesions at the front of the cell moved backward in unison (arrowhead) and generated significant ECM deformation. See also movie 3 at jcs.biologist.org/supplemental. At the same time, cell body adhesions moved forward (arrow), resulting in contractile-like shortening of the cell and ECM compression at the base of lamellipodia. See also movies 3 and 4 at jcs.biologist.org/supplemental.

View larger version (34K): [in a new window]   Fig. 5. Quantitative comparison of the magnitude and direction of the movement of adhesions and nearby ECM landmarks. (A,B) Displacement data for two different cells undergoing extension. A significant correlation between adhesion and ECM displacement was found for every adhesion that was tracked (data from each adhesion are shown in a different color). However, the slope of the relationship varied from one adhesion to the next. (C) The pooled data for all five extensions analyzed. A highly significant correlation was found, but the magnitude of adhesion movement was larger than that of the ECM. (D) Data from a cell that underwent slow lamellipodial retraction. A high correlation between the magnitude of ECM and adhesion displacements (R=0.97, P<0.001), and the slope was much closer to 1.0. (E) Comparison of the direction of adhesion and ECM movements for all six experiments combined (five extensions and one retraction). A high correlation was demonstrated, and the slope was 1.0. (F) Since the adhesions tended to assume an oblong shape, we also measured the angle of the long axis of each adhesion (adhesion orientation). The adhesion orientation was highly correlated with the movement of the ECM.

View larger version (96K): [in a new window]   Fig. 6. Effects of cytochalasin D on living corneal fibroblasts plated on top of a fibrillar collagen matrix. The red tracks show ECM displacements, and GFP-zyxin is shown in green. (A) A cell after 140 minutes of time-lapse imaging in complete media. (B) Within three minutes after adding cytochalasin D to the media, cell elongation and ECM relaxation were observed (red tracks). This was most dramatic near the front of the cell. Although the cell body moved forward, decompression of the ECM behind the lamellipodium was still observed (compare length of arrows in A and B). Coincident with ECM relaxation, the intensity and clarity of the adhesions was significantly reduced. (C) After reperfusion with complete media, the adhesions reformed and the ECM was pulled back in (yellow tracks, and arrow). Bar, 10 µm.