Cooperative effects of Rho and mechanical stretch on stress fiber organization

Abstract

The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rhokinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2⌬1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional Ϸ3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2⌬1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.cytoskeletal dynamics ͉ endothelial cells ͉ mechanotransduction ͉ Rho-kinase T he tension generated by contraction of adherent cells against their underlying surface results in an internal stress field that depends on the organization of the cytoskeleton and the associated adhesive contacts (see ref. 1 for review). Intracellular forces have an important role in cellular functions such as migration, proliferation, apoptosis, differentiation, and gene expression (see refs. 2-4 for reviews). Actin stress fibers, which are formed in response to cell contraction (5), consist of bundles of actin microfilaments cross-linked by ␣-actinin, myosin, myosin light-chain, tropomyosin, and other proteins arranged in a manner similar to that in muscle sarcomeres (6). Stress fibers represent the main contractile apparatus in non-muscle cells (7) and are the primary structures associated with intracellular tension. Stress fibers terminate at focal adhesions, which attach the cell to the extracellular matrix (8). Isometric contraction of a cell would result in tension development in the stress fibers, which are anchored at their ends.The activation of the small GTPase Rho leads to stress fiber assembly (9) and cell contraction by means of myosin light chain phosphorylation (5), which is regulated by Rho-kinase, a downstream effector of Rho (10). mDia, another Rho effector, is also involved in stress fiber formation downstream of Rho activation (11), possibly by regulating actin polymerization and focal adhesion turnover through its association with profilin (12, 13) and src-tyrosine-kinase (14), respectively.Cyclic uniaxial stretch induces the orientation of stress fibers in endothelial cells ...

A thermodynamically motivated model for stress-fiber reorganization

“…(ii) Cells "sense" the stiffness of their substrates and exert smaller tractions on more compliant substrates (Discher et al 2005). (iii) When substrates on which cells are cultured are subjected to cyclic strains, cells "avoid strain" such that stress-fibers form in the direction perpendicular to the imposed cyclic strain (Kaunas et al 2005). On the other hand, when cells cultured in three-dimensional (3D) tissues are subjected to cyclic strain, "strain alignment" is observed such that stress-fibers now form in the direction of the imposed cyclic strain (Nieponice et al 2007)…”

“…In these experiments the cytoskeletal network formed perpendicular to the imposed cyclic strains and this was interpreted as strain avoidance (Buck 1980). Typically this avoidance increased with increasing imposed strain amplitude at a fixed frequency of loading (Kaunas et al 2005). This was interpreted as a sensitivity to imposed strain-rate (Wei et al 2008).…”

“…Experiments regarding cytoskeletal arrangements in response to cyclic mechanical stimuli were initially performed on cells seeded on two-dimensional substrates that were stiff relative to the cells (Neidlinger-Wilke et al 2001;Kaunas et al 2005). In these experiments the cytoskeletal network formed perpendicular to the imposed cyclic strains and this was interpreted as strain avoidance (Buck 1980).…”

Prediction of Cell Alignment on Cyclically Strained Grooved Substrates

“…For instance, cells orient perpendicular to cyclic strain when cultured on stiff substrates or on biaxially constrained collagen gels (1)(2)(3)(4)(5)(6)(7). This phenomenon, called strain avoidance (SA), depends on the profile of the cyclic strain because the degree of cellular (re)orientation is proportional to the amplitude and frequency of the strain (1,2).…”

“…In fact, cellular alignment generally corresponds to the main SF direction (2), and experiments have shown that these acto-myosin bundles are essential for the reorientation of cells in response to cyclic strain (3,5). On the other hand, FAs are crucial to sense and interact with topographical stimuli, because they form the link between cells and their surroundings (34).…”