Time-lapse DIC images of a corneal fibroblast plated inside a 3-D collagen matrix taken at a focal plane near the middle of the cell. DIC imaging allowed detailed visualization of the fibrillar collagen organization. Individual collagen fibrils are easily discerned adjacent to the cell (arrows in frame 1), and movement and realignment of the fibrils can be directly observed. Extension of pseudopodial processes generated tractional forces on the ECM as indicated by pulling in of the collagen fibrils in front of the cell (left). At the same time, the cell body and adjacent ECM moved forward. Localized compression of the ECM along a pseudopodia was observed (circled region at end of movie). The time given is referenced to the start of time-lapse imaging.
Time-lapse color overlay images of corneal fibroblast (green = GFP-zyxin, red = DIC). The cell is shown extending pseudopodial processes at both ends. In general, the focal adhesion movements appeared to correlate with the ECM deformation observed by DIC. Interestingly, contractile shortening and matrix compression appeared to take place at the ends of the cell, resulting in stretching of the matrix near the middle of the cell body (arrows in last frame). The time given is referenced to the start of time-lapse imaging.
GFP-α-actinin transfected corneal fibroblast (white) two days after plating inside collagen matrix surrounded by FEM strain maps generated using ANSYS, showing regions of matrix tension (red and orange) and compression (blue). Stress on the matrix in serum containing media is reduced when the cell is switched to Y-27632. Stress is re-established after switching back to Serum. Note that the clarity of the stress fibers is reduced following incubation with Y-27632.
Cell-matrix mechanical interactions in response to PDGF. Tracking of the ECM displacements showed minimal collagen displacement prior to the addition of PDGF (red tracks, crosses mark position at time 0:00). However, following addition of PDGF, the matrix in front of the cell was pulled inward by the extending pseudopodial processes, resulting in compression of the ECM.
Color overlay of GFP-zyxin (green) and collagen fibils (red) shown over a range of projection angles for a cell cultured for 24 hours in Serum containing media. Note that there is compaction and alignment of collagen fibrils associated with the GFP-zyxin labeled focal adhesions. Collagen fibrils were imaged using confocal reflection microscopy.
Color overlay of f-actin (green) and collagen fibils (red) shown over a range of projection angles for a cell cultured for 24 hours in serum containing media. The cell has a bipolar morphology. Note that there is compaction and alignment of collagen fibrils parallel to stress fibers at the end of pseudopodia, but little collagen reorganization along the cell body.
Color overlay of f-actin (green) and collagen fibils (red) shown over a range of projection angles for a cell cultured for 24 hours in serum containing media containing the Rho kinase inhibitor Y-27632. Cell has a more 3-dimensional dendritic morphology than cells in serum alone. No consistent pattern of matrix compaction and alignment is observed.