Dynamic assessment of fibroblast mechanical activity during Rac-induced cell spreading in 3-D culture

Abstract

The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 microm thick fibrillar collagen matrices and cultured for 1-2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent.

Figure 1

Rac activation data. Graph shows the mean and standard deviation of four independent experiments. Data shown is luminescence over background signal (wells incubated with lysis buffer alone instead of cell lysates), normalized to 1 ng Rac1 protein controls. In all four experiments, both 10 and 30 minutes exposure to PDGF increased the level of Rac activation as compared to untreated cells; this increase reached statistical significance at 30 minutes (P < 0.05, Repeated Measures ANOVA, with Holm-Sidak method for multiple comparisons).

Figure 2

PDGF-induced cell spreading. A–C. HTK cell plated inside a 3-D collagen matrix, cultured for 24 hours in serum-free media, and transferred to the microscope stage. DIC imaging allowed detailed visualization of pseudopodial processes (arrows) and the individual collagen fibrils surrounding them. No significant changes in cell morphology were observed in basal media (compare A and B). However, addition of PDGF induced rapid cell spreading via the extension of existing pseudopodial processes and the formation of new processes (C). Time shown is hours:minutes since the start of time-lapse imaging. D–F. A second HTK cell showed similar spreading over a longer time course. G–H. Quantitative analysis (n = 16 cells) demonstrated an increase in projected cell length, projected cell area, and the number of pseudopodial processes following PDGF treatment.

Figure 3

Cell-matrix mechanical interactions in response to PDGF. A–C. Cell-induced displacement and realignment of collagen fibrils was observed during PDGF-induced spreading. Tracking of the ECM displacements showed minimal collagen displacement prior to the addition of PDGF (B, 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 (C), resulting in compression of the ECM (yellow arrows). D–G. Higher magnification assessment of cellular interactions with individual collagen fibrils. PDGF was added at 1:04. In this example, a collagen fibril (arrowheads) in front of an extending process (arrows) is aligned somewhat parallel to the direction of spreading (D). The extending process engages the fibril (E), pulls it into alignment (F), then continues to spread along it (G). This results in alignment of the collagen fibril parallel with the pseudopodia. H–L. Cellular interactions with two collagen fibrils (arrowheads) aligned somewhat perpendicular to the direction of spreading. PDGF was added at 0:26 minutes. The extending process (arrows) engages the first fibril (I), pushes past it to engage the second fibril (J), then pulls the fibrils together (K). This results in compaction of the collagen fibrils in a direction perpendicular to the extending process (L; yellow lines indicate initial position of collagen fibrils, red lines indicate final position of collagen fibrils).

Figure 4

Color overlays of GFP-zyxin (green) and DIC (red). PDGF was added at 0:40 minutes. Formation of cell-matrix adhesions at the leading edge of an extending pseudopodia was associated with centripetal displacement and/or bending of the collagen fibrils with which it interacted (B and C, arrows). Cytochalasain D induced disassembly of adhesions and matrix relaxation (E).

Figure 5

The role of ROCK on the subcellular pattern of force generation and cell-matrix interactions in response to PDGF. A–C. Perfusion was changed to PDGF at 0:40 (hours:minutes), and PDGF + Y-27632 at 1:40. Addition of the ROCK inhibitor Y-27632 (10μM) to PDGF treated cells induced additional cell spreading and elongation. Cells also assumed a more convoluted shape with thinner cell processes (arrows), suggesting a reduction of cellular tension. D–F. Following 30 minutes of ROCK inhibition, Y-27632 was washed out by switching the perfusion back to PDGF alone. Cell processes (arrows) became thicker (compare D and E), and increased cellular forces were observed, particularly at the base of pseudopodial processes (E, yellow arrows). Subsequent treatment with cytochalasin D resulted in elongation and thinning of cellular processes, and ECM decompression (F, yellow arrow). G–K. Cell matrix interactions at the end of a pseudopodia following ROCK inhibition. A thin dendritic process (arrows) branches off from another process. The branching process extends along a collagen fibril, engages a second fibril which is oriented perpendicular (arrowhead), and displaces this fibril inward (K, yellow line indicates initial fibril position, red line indicates final fibril position). L–N. The effect of PDGF following pre-incubation with Y-27632. Addition of Y-27632 to basal media induced cell elongation and relaxation of tractional forces (M, red tracks, crosses mark position at time 1:41), indicating that there is a basal level of ROCK activity in serum-starved corneal fibroblasts. Subsequent addition of PDGF induced additional elongation, and significant pseudopodial ruffling and branching of cell processes (N), resulting in a more dendritic appearance. Large tractional forces were not observed (M, red tracks, crosses mark position at time 2:42). Perfusion with Y-27632 (100 μM) began at 1:41, and perfusion with Y-27632 plus PDGF at 2:42.

Figure 6

The role of myosin II on the subcellular pattern of force generation and cell-matrix interactions in response to PDGF. A–C. Perfusion was changed to the non muscle myosin II inhibitor blebbistatin (100 μM) at 0:54 (hours:minutes), and PDGF + blebbistatin at 2:18. Treatment of cells with blebbistatin resulting in significant relaxation of tractional forces (B, red tracks, crosses mark position at time 0:54). Subsequent addition of PDGF stimulated additional cell spreading, without the generation of large tractional forces (C, red tracks, crosses mark position at time 2:18). D–F. Perfusion was changed to the non muscle myosin II inhibitor blebbistatin at 1:00 (hours:minutes), and PDGF + blebbistatin at 2:10. Addition of PDGF in the presence of blebbistatin also induced extensive branching and pseudopodial ruffling (E, arrows), resulting in a dendritic cell morphology (F). G–K. Cell matrix interactions at the end of a pseudopodia during cell spreading following myosin II inhibition. A thin dendritic process (arrow) branches off from another process. The branching process engages fibril (arrowhead), and displaces this fibril inward (K, yellow line indicates initial fibril position, red line indicates final fibril position). Perfusion with blebbistatin began at 0:55, and perfusion with blebbistatin plus PDGF at 2:10.