Traction forces of fibroblasts are regulated by the Rho-dependent kinase but not by the myosin light chain kinase

Abstract

Adhesive cells show complex mechanical interactions with the substrate, however the exact mechanism of such interactions, termed traction forces, is still unclear. To address this question we have measured traction forces of fibroblasts treated with agents that affect the myosin II-dependent contractile mechanism. Using the potent myosin II inhibitor blebbistatin, we demonstrate that traction forces are strongly dependent on a functional myosin II heavy chain. Since myosin II is regulated by both the myosin light chain kinase (MLCK) and, directly or indirectly, the Rho-associated kinase (ROCK), we examined the effects of inhibitors against these kinases. Interestingly, inhibition of the myosin light chain kinase had no detectable effect, while inhibition of the Rho-dependent kinase caused strong inhibition of traction forces. Our results indicate that ROCK and MLCK play non-redundant roles in regulating myosin II functions, and that a subset of myosin II, regulated by the Rho small GTPase, may be responsible for the regulation of traction forces in migrating fibroblasts.

Figure 1. Inhibition of traction force production by a myosin II inhibitor

Phase images of an NIH3T3 fibroblast on collagen-coated polyacrylamide substrate are shown before (A), and after (B), treatment with 10 μM blebbistatin for 30 minutes. Vector plots show the corresponding traction stress before (C), and after (D), the treatment. Note the presence of lamellipodium despite the strong inhibition of traction forces. Bar, 10 μm.

Figure 2. Decrease of MRLC phosphorylation after the treatment with inhibitors

Lysates of NIH3T3 fibroblasts, treated with medium alone (lane 1), 10 μM Y-27632 for 30 (lane 2) or 60 (lane 3) minutes, 20 μM BATI inhibitory peptide for 30 (lane 4) or 60 (lane 5) minutes, 10 μM ML-7 for 30 (lane 6) or 60 (lane 7) minutes, 50 μM ML-7 for 30 (lane 8) or 60 (lane 9) minutes at 37°C are subjected to immnoblotting. Upper lanes are probed with anti-actin antibodies, as a loading control, lower lanes are probed with anti-monophospho-MRLC antibodies. Arrow in the lower panel indicates the position of MRLC. The anti-phospho-MLCK antibody also reacts with a band of slightly higher molecular weight (asterisk), which likely represents an MRLC isoform [44]. Y-27632 causes a rapid but partial inhibition of MRLC phosphorylation, which shows no apparent change beyond 30 minutes of incubation. In contrast, the effect of BATI does not become clear until 60 minutes in this assay. Treatment with 10 μM ML-7 causes little inhibition of MRLC phosphorylation even after prolonged incubation, while treatment with 50 μM ML-7 shows a rapid, strong inhibition.

Figure 3. Lack of effects of the inhibition of MLCK activity on traction forces

Phase images of an NIH3T3 fibroblast on collagen coated polyacrylamide substrate are shown before (A), and after (B), treatment with 20 μM BATI inhibitory peptide for 30 minutes. Vector plots show the corresponding traction stress before (C), and after (D), the treatment. Bar, 10 μm.

Figure 4. Inhibition of traction force production by a ROCK inhibitor

Phase images of an NIH3T3 fibroblast on collagen coated polyacrylamide substrate are shown before (A), and after (B), treatment with 20 μM Y-27632 for 30 minutes. Vector plots show the corresponding traction stress before (C), and after (D), the treatment. Note the presence of lamellipodium despite the strong inhibition of traction forces. Bar, 10 μm.

Figure 5. Disruption of the organization of actin and vinculin by agents that affect myosin II

NIH 3T3 fibroblasts treated with 10 μM blebbistatin (C, D), 20 μM BATI peptide (E, F), 20 μM Y-27632 (G, H), or carrier solution alone (A, B), for 30 minutes are double stained with fluorescent phalloidin (A, C, E, G) and antibodies against vinculin (B, D, F, H). Both blebbistatin and Y-27632 cause disappearance of stress fibers and focal adhesions, although cells in Y-27632 show a concentrated band of vinculin along the cell periphery. BATI induces no apparent effect on stress fibers, and a subtle change in the appearance of focal adhesions. Bar, 20μm.

Figure 6. Effects of MLCK inhibition on cell-substrate adhesions

IRM images are shown before (A, B), and after (C, D, E), treatment with 20 μM BATI. Note the persistence of very dark adhesion plaques, the formation of dark patches of close contact near the leading edge, and the continuous forward migration of the cell. Numbers indicate minutes after treatment. Bar, 20 μm.

Figure 7. Effects of ROCK inhibition on cell-substrate adhesions

IRM images are shown before (A, B), and after (C-H), treatment with 20 μM Y-27632. Note the dramatic disappearance of most adhesion plaques near the leading edge upon prolonged treatment, while the cell expands and takes an abnormal shape. Numbers indicate minutes after treatment. The line in E-H serves as the reference for the visualization of shape change and the identification of residual adhesion plaques. Bar, 20 μm.