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

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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.

Section snippets

Cell culture, treatments, and immunoblotting

NIH-3T3 mouse embryonic fibroblasts were purchased from ATTC. Cells were maintained in DMEM supplemented with 10% donor calf serum (Hyclone), 50 U/ml penicillin, 50 μg/ml streptomycin and 2 mM l-glutamine (Gibco, Grand Island, NY). Pharmaceutical reagents purchased from commercial sources include ML-7 (an MLCK inhibitor [23]; Calbiochem, San Diego, CA), blebbistatin (a non-muscle myosin II inhibitor [8]; Toronto Research, Toronto, Canada), Y-27632 (a ROCK inhibitor [24]; Mitsubishi Pharma, Osaka,

Myosin II plays a crucial role in generating traction forces

To assess the contribution of non-muscle myosin II motor activities to the production of traction forces, we applied traction force microscopy to NIH 3T3 fibroblasts treated with the inhibitor blebbistatin. Treatment with 10 μM blebbistatin for 30 min caused strong inhibition of traction forces from an average of 0.084 ± 0.04 dynes/cm2 to the noise level of 0.0065 ± 0.002 dynes/cm2 (Fig. 1). These results support the notion that myosin II produces most if not all of traction forces. However, consistent

Discussion

Generation of strong traction forces represents a common function of adhesive cells including fibroblasts, epithelial cells, endothelial cells, and macrophages [37]. However, despite the advances in detection, the mechanism for the production and regulation of these forces has remained elusive. In this study we have examined the involvement of myosin II in force generation in cultured fibroblasts, using traction force microscopy in conjunction with a cache of small molecule and peptide

Acknowledgments

Except for immunoblotting, experimental results were collected in the laboratory of Y.-L.W. through the support of NIH Grant GM-32476. K.A.B. was responsible for experiments on the effects of ROCK on traction forces and for the preparation of the manuscript. K.H. performed the immunoblotting experiment. M.D. was responsible for computational analysis of traction stress, supported by NIH Grant R01 GM-61806. Y.L.W. was responsible for the general conceptualization and supervision of the project.

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