Cell locomotion and focal adhesions are regulated by substrate flexibility

Citation:

ROBERT J. PELHAM, JR., AND YU-LI WANG
Department of Physiology, University of Massachusetts Medical School, Worcester Foundation Campus, 222 Maple Avenue, Shrewsbury, MA 01545
Edited by Thomas D. Pollard, Salk Institute for Biological Studies, La Jolla, CA, and approved October 16, 1997 (received for review June 4, 1997)

Summary:

Response of rat kidney epithelial and 3t3 fibroblast cells was examined with respect to varying substrate elasticity of collagen-coated polyacrylamide gels. The gels were created on glass slides that were intensively sterilized and treated with nitrocellulose to promote binding of the polyacrylamide. The substrate stiffness was changed by forming gels of a constant concentration of 10% acrylamide, while varying the concentration of bis-acrylamide from 0.03 to 0.26%, which promotes cross-linking and increases the elasticity of the gel. After washing and photoactivation, a solution of type I collagen was layered onto the substrated and allowed to react ovbernight. The stiffness of the substrate was tested macroscopically and microscopically by deformation of glass microneedles. Force and resulting strain of a sample of known cross-sectional area were used to calculate Young's modulus. The elastic modulus of the 0.26% polyacrylamide gel showed a 12 fold increase over that of the 0.03% gel in macroscopic tests, and a 16 fold difference in compliance in microscopic tests.



After the cells were cultured in the different substrates using identical media, cell migration was measured using time-lapse capture by phase-contrast microscope. On rigid substrates, both the ERK and the 3t3 cells exhibited flat, adherent morphology. On softer substrates, the ERK cells became less well-spread and exhibited greater ruffling of the membrane. 3T3 cells appeared more spindle-shaped and lost most of their stress fibers. 3T3 migration was increased in the less rigid sample, while the ERK cells tended to colonize and did not differ significantly in term of migration. To ensure the differences in cell morphology were not due to differences in collagen concentration induced by subtrate differences, the cells were lysed and then the subtrate was immunostained for collagen. This resulted in almost identical concentrations of collagen between the samples.



Using microinjections of fluorescent vinculin, cells cultured on more deformable substrates assumed irregularly-shaped focal adhesion sites, that appeared and disappeared over a period of 10 minutes. Focal adhesion sites in cells cultured in stiffer substrates persisted in the same location. Immunostaining for phosphotyrosine, which are found extensively in focal adhesion sites, showed similar morphological changes between the focal adhesion sites of cells cultures on soft vs stiff substrates. Immunoblot studies indicated greater overall concentration of phosphotyrosine in cells on stiffer substrate. To further corroborate the relationship between tyrosine phosphorylation and focal adhesion sites, a tyrosine phosphatase inhibitor was added to the soft-substrate culture. This induced a focal adhesion site morphology similar to that of the stiff-substrate culture. To highlight the role of the cell cytoskeleton in forming coherent focal adhesion shapes, a myosin light chain kinase inhibitor was added, to the effect of disrupting the regular focal adhesion site shape.



Significance:

Adhesions between cells and the extracellular matrix (ECM) are known to modulate numerous critical cellular events such as gene expression, embryonic development, and cell locomotion. This process involves interactions of ECM proteins such as collagen, fibronectin, or vitronectin, with the integrin family of transmembrane receptors. Subsequent cascade of events include the phosphorylation of proteins at adhesion sites and the recruitment of various cytoskeletal proteins to form focal adhesions. These findings are very relevant to the possible role of substrate stiffness on the differentiation of stem cells and may point to the pathway by which differentiation is induced.

A number of observations suggest that cell adhesion reactions involve not only receptor binding but also physical interactions and the cytoskeleton. According to previous findings ECM proteins must be immobilized or cross-linked in order to illicit activation of signaling pathways. In addition, previous studies have shown that cells can respond to forces exerted through surrounding fluid, adhered beads, or substrates. Thus, mechanical forces may play a role in adhesion responses, and, conversely, cells may actively probe and respond to mechanical characteristics of the surrounding environment.

Criticism:

It is a very well-thought out experiments that took into account confounding factors such as the possible variations in collagen concentration due to the difference degrees of cross-linking between the substrates of different bis-acrylamide concentrations. The careful selection of kinases inhibitors and phosphatase inhibitors showed a clear and well-developed initial theory of the mechanism that relates integrin- substrate interactions to changes in focal adhesion site morphology, to the changes in cell morphology and migration. The two types of tensile tests for substrate elasticity, demonstrates that the researchers realized that although the elastic modulus may be easily taken from macroscopic tests, the cells only interact with a small part of the substrate: the surface. Therefore, it was good to approximate the substrate response to cells by a probe on the microscopic scale, by deformation of a glass microneedle. However, even the microneedle does not truly take into account the deformation characteristics of individual collagen fibrils, which are theoretically what the cell integrins attach to. Due to the non-linear elastic behavior of collagen and other structural proteins, it is difficult to say if the elastic response of the acrylamide substrate is directly translated to the cells.

However, some facts may have been overlooked, such as the clumping tendencies of the kidney epithelial cells that prevented relevant data on migration from being taken. Another significant difference between the experimental set-up and the molecular event it is modeling is the fact that cellular interactions occur in a fully 3-D environment. The alignment of the cytoskeleton may change significantly in a 3-D environment that affords attachment points in all directions. It may be that the cell may become more sensitive to changes in elasticity in 3-D vs a 2-D substrate. Although the pros of the 2-D culture, such as ease of access to the cells, may outweigh the variation afforded by the 3-D culture.