Reference: Biol. Bull. 194: 348-350. (June, 1998) Cell Locomotion and Focal Adhesions Are Regulated by the Mechanical Properties of the Substrate ROBERT J. PELHAM JR. AND YU-LI WANG Department of Physiology, University of Massachusetts Medical School, Worcester Foundation Campus, 222 Maple Ave, Shrewsbury, Massachusetts 01545 Cell-cell adhesion and cell-substrate adhesion are im- portant interactions that modulate intracellular signaling pathways, as well as various cellular events from gene expression to cell locomotion (Juliano and Haskill, 1993). The full response to adhesion seems to involve not only the cross-linking of integrins but also mechanical input through these receptors (Craig and Johnson, 1996; Wang et al., 1993; Ingber, 1993; Chrzanowska-Wodnika and Burridge, 1996; Choquet et al., 1997). To explore this possibility, we have examined the motility and cytoskele- tal organization of NRK epithelial cells and 3T3 fibro- blasts cultured on substrateshaving varying mechanical properties (Pelham and Wang, 1997). Flexible, optically clear substrates were prepared by covalently linking type I collagen to polyacrylamide sheets. The flexibility of the substrate was manipulated by maintaining the acrylamide concentration at 10% while varying the bis-acrylamide contents between 0.03% and 0.26%. In this manner, we were able to maintain a con- stant chemical environment regardless of substrate flexi- bility. The Young’s Modulus of the substrate, determined by measuring the extent of stretching in response to known applied forces, showed a 13-fold difference be- tween sheets of 0.26% and 0.03% bis-acrylamide. When probed microscopically with a calibrated microneedle, the substrates showed 16-fold difference in compliance (-7.3 x lo-’ newtons/pm versus -4.6 X lop8 newtonslym). On more rigid substrates, both NRK epithelial cells and 3T3 fibroblasts were well spreadand appearedindis- tinguishable from those cultured on glass or plastic sur- This paper was originally presented at a workshop titled The Cytoskel- eton: Mechanical, Physical, and Biological Interactions. The workshop, which was held at the Marine Biological Laboratory, Woods Hole, Massachusetts, from 15-17 November 1996, was sponsored by the Center for Advanced Studies in the Space Life Sciences at MBL and funded by the National Aeronautics and Space Administration under Cooperative Agreement NCC 2-896. faces. However, when cells were cultured on increasingly flexible substrates, there was a corresponding change in morphology: NRK cells became less well spread and ir- regularly shaped. Highly active, phase-dense ruffles ap- peared, not only along the periphery, but on the ventral surface of the cell. 3T3 cells lost most of their stress fibers and became increasingly spindle-shaped,with a concomi- tant increase in the rate of locomotion. These observations suggest that cellular motility is reg- ulated according to the mechanical properties of the sur- rounding environment. The initial response likely origi- nates at cell-substrate adhesion sites, where mechanical input can be translated into intracellular signals. Indeed, microinjection of fluorescent vinculin showed that focal adhesions in cells cultured on highly flexible substrates become irregular in morphology and much less stable than those in cells on rigid substrates.Similar differences were found when cells were immunostained for phospho- tyrosine. In addition, immunoblots with anti-phosphotyro- sine Py20 antibody indicated that the overall extent of phosphorylation on flexible substrates was greatly re- duced in comparison with that of cells plated on plastic or rigid surfaces. Treatment of cells on flexible substrates with phenylar- sine oxide, a tyrosine phosphataseinhibitor, resulted in the formation of normal focal adhesions. Conversely, treatment of cells on firm substrateswith myosin II/myo- sin light chain kinase inhibitors 2,3-butanedione monox- ime or KT5926 causedlarge focal adhesions to disappear, as on flexible substrates. These results are consistent with the idea that cells use an actin-myosin-based push-pull mechanism at their integrin receptors to probe their me- chanical environment. The responses then affect the level of tyrosine phosphorylation on the cytoplasmic side, which in turn regulates the formation and stability of focal adhesionsand possibly motile activities (Fig. 1). The present results, together with a growing list of observations suggestingthat cells can respond to both the 348